HIV-1 GP140 immunogens comprising modified NHR1 regions that stabilize pre-fusion envelope conformations

ABSTRACT

The present invention provides HIV-1 vaccine immunogens. Some of the immunogens contain a soluble gp140-derived protein that harbors a modified N-terminus of the HR1 region in gp41. Some of the immunogens contain an HIV-1 Env-derived trimer protein that is presented on a nanoparticle platform. The invention also provides methods of using the HIV-1 vaccine immunogens for eliciting an immune response or treating HIV infections.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a divisional of U.S. patent application Ser.No. 16/780,495 (filed Feb. 3, 2020; now pending), which is a divisionalof U.S. patent application Ser. No. 16/176,200 (filed Oct. 31, 2018; nowU.S. Pat. No. 10,647,748), which is a continuation-in-part of PCT PatentApplication No. 2017/030375 (filed May 1, 2017; now expired), whichclaims the benefit of priority to U.S. Provisional Patent ApplicationNo. 62/330,604 (filed May 2, 2016). The full disclosures of the priorityapplications are incorporated herein by reference in their entirety andfor all purposes.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under grant numbersAI100663, AI084817, GM094586 and AI110657 awarded by the NationalInstitutes of Health and grant number DE-AC02-06CH11357 awarded by theU.S. Department of Energy. The government has certain rights in theinvention.

BACKGROUND OF THE INVENTION

Human immunodeficiency virus type 1 (HIV-1) is the primary cause of theacquired immune deficiency syndrome (AIDS) which is regarded as one ofthe world's major health problems. It is an RNA virus of the familyRetroviridae. The HIV-1 genome encodes at least nine proteins which aredivided into three classes: the major structural proteins Gag, Pol andEnv, the regulatory proteins Tat and Rev, and the accessory proteinsVpu, Vpr, Vif and Nef. HIV-1 can be divided into several differentclades, for example A, B, C, D, E, F, G, H, J and K, which vary inprevalence throughout the world. Each clade comprises different strainsof HIV-1 which have been grouped together on the basis of their geneticsimilarity.

The initial phase of the HIV-1 replicative cycle involves the attachmentof the virus to susceptible host cells followed by fusion of viral andcellular membranes. These events are mediated by the exterior viralenvelope glycoproteins which are first synthesized as afusion-incompetent precursor envelope glycoprotein (Env) known as gp160.The genetic diversity of HIV-1 renders extremely difficult for thedevelopment of an effective vaccine against strains from multiple HIV-1clades. Tremendous efforts have been expended in the past two decades toproduce a preventive HIV vaccine. While several candidate vaccines havebeen developed, they all failed to prevent HIV-1 infection in clinicaltesting.

The generation of an antibody response capable of neutralizing a broadrange of clinical isolates remains a major challenge in humanimmunodeficiency virus type 1 (HIV-1) vaccine development. There is astrong and urgent need for a vaccine that that is safe and efficaciousaround the world. The present invention addresses this and other unmetneeds in the art.

SUMMARY OF THE INVENTION

In one aspect, the invention provides modified HIV-1 envelope gp140proteins. The proteins are composed of a gp120 polypeptide and a gp41polypeptide, with the N-terminus of heptad 1 region (HR1) of the gp41polypeptide being replaced by a loop sequence of about 6 to about 14amino acid residues in length that stabilizes the pre-fusion gp140structure. In some of these proteins, the gp41 polypeptide isgp41_(ECTO). Preferably, the modified HIV-1 gp140 protein is a trimer.In some embodiments, the gp120 polypeptide and the gp41 polypeptide arederived from the same HIV-1 strain or subtype. For example, both thegp120 polypeptide and the gp41 polypeptide in the modified HIV-1 gp140protein can be derived from HIV-1 strain BG505. In some embodiments, thegp120 polypeptide and the gp41 polypeptide are derived from differentHIV-1 strains or subtypes. For example, an engineered gp41 polypeptidefrom HIV-1 strain BG505 as exemplified herein can be used to formchimeric gp140 immunogens with a gp120 polypeptide derived from manyother HIV-strains or subtypes.

In some modified HIV-1 envelope gp140 proteins of the invention, theloop sequence contains (GS)n (SEQ ID NO:23), with n being any integerbetween 3 and 7, inclusive. In some of these embodiments, the loopsequence is (GS)₄ (SEQ ID NO:24). In some embodiments, the loop sequenceis obtained via rational redesign. In some of these embodiments, theloop sequence is obtained by ensemble-based protein design. In somemodified HIV-1 gp140 proteins of the invention, the loop sequencecontains 10 amino acid residues. Some examples of these loop sequencesare shown in SEQ ID NOs:1-5. In some other modified HIV-1 gp140proteins, the loop sequence contains 8 amino acid residues. Someexamples of these loop sequences are shown in SEQ ID NOs:6-10.

In some embodiments, the modified HIV-1 gp140 proteins of the inventionfurther contain a flexible linker sequence that substitutes for thecleavage site sequence between gp120 and gp41. In some of theseembodiments, the linker sequence has a sequence of (G₄S)₂ (SEQ ID NO:22)or SGS, which substitutes for residues 508-511 at the cleavage site. Insome other embodiments, the linker sequence contains 8 amino acidresidues and substitutes for residues 501-518 at the cleavage site. Inthese embodiments, numbering of the amino acid residues corresponds tothat of HIV-1 strain BG505. SOSIP.664 gp140. In some exemplifiedproteins, the linker sequence contains the sequence shown in any one ofSEQ ID NOs:16-20.

In some embodiments, the modified HIV-1 gp140 proteins of the inventionfurther contains (a) an engineered disulfide bond between gp120 and gp41and/or (b) a stabilizing mutation in gp41. In some of these embodiments,the engineered disulfide bond is between residues A501C and T605C, andthe stabilizing mutation is I559P.

Some modified HIV-1 gp140 proteins of the invention contain a gp140trimer derived from HIV-1 strain BG505, with each gp140 monomercontaining a gp120 polypeptide and a gp41_(ECTO) polypeptide, and theN-terminus of heptad 1 region (HR1) (SEQ ID NO:28) in gp41_(ECTO)polypeptide being replaced with a loop sequence shown in SEQ ID NO:6. Insome of these embodiments, the protein additionally contains (a) alinker sequence (G₄S)₂ (SEQ ID NO:22) that substitutes for residues508-511 at the cleavage site, and (b) an engineered disulfide bondbetween residues A501C and T605C.

In another aspect, the invention provides HIV-1 vaccine immunogens thatcontain a modified trimeric HIV-1 envelope gp140 protein. In theseimmunogens, the gp140 protein contains a gp120 polypeptide and agp41_(ECTO) polypeptide, with the N-terminus of heptad 1 region (HR1) ofthe gp41_(ECTO) polypeptide being replaced with a loop sequence of about6 to about 14 amino acid residues that stabilizes the pre-fusion gp140structure. In some embodiments, the loop sequence contains (GS)n (SEQ IDNO:23), with n being any integer between 3 and 7, inclusive. In some ofthese embodiments, the loop sequence has a sequence of (GS)₄ (SEQ IDNO:24). In some embodiments, the loop sequence is obtained via rationalredesign, e.g., by ensemble-based protein design. In some embodiments,the loop sequence contains 10 amino acid residues, e.g., any sequence asshown in SEQ ID NOs:1-5. In some other embodiments, the loop sequencecontains 8 amino acid residues, e.g., a sequence as shown in any one ofSEQ ID NOs:6-10.

Some HIV-1 vaccine immunogens of the invention additionally contain aflexible linker sequence that substitutes for the cleavage site sequencebetween gp120 and gp41_(ECTO). In some of these embodiments, the linkersequence contains (G₄S)₂ or SGS, and substitutes for residues 508-511 atthe cleavage site. In some embodiments, the linker sequence contains 8amino acid residues and substitutes for residues 501-518 at the cleavagesite. In these embodiments, numbering of the amino acid residuescorresponds to that of HIV-1 strain BG505. SOSIP.664 gp140. In someembodiments, the linker sequence contains a sequence as shown in any oneof SEQ ID NOs:16-20.

Some HIV-1 vaccine immunogens of the invention additionally contain anengineered disulfide bond between gp120 and gp41. In some of theseembodiments, the engineered disulfide bond is between residues A501C andT605C. Some of the HIV-1 vaccine immunogens contain a gp140 trimerderived from HIV-1 strain BG505, with each gp140 monomer containing agp120 polypeptide and a gp41_(ECTO) polypeptide, and with the N-terminusof heptad 1 region (HR1) (SEQ ID NO:28) in gp41^(ECTO) polypeptide beingreplaced with a loop sequence shown in SEQ ID NO:6. In some embodiments,the modified HIV-1 gp140 protein further contains (a) a linker sequence(G₄S)₂ (SEQ ID NO:22) that substitutes for residues 508-511 at thecleavage site, and (b) an engineered disulfide bond between residuesA501C and T605C.

In another aspect, the invention provides HIV-1 vaccine compositionsthat contain an HIV-1 Env-derived trimer immunogen presented on aself-assembling nanoparticle or a virus-like particle (VLP). In some ofthese embodiments, the HIV-1 Env-derived trimer immunogen is V1V2, gp120or gp140. In some embodiments, the HIV-1 Env-derived trimer immunogen isa modified gp140 protein that contains a gp120 polypeptide and agp41_(ECTO) polypeptide that has the N-terminus of heptad 1 region (HR1)of the gp41_(ECTO) polypeptide being replaced with a loop sequence ofabout 6 to about 14 amino acid residues that stabilizes the pre-fusiongp140 structure.

In some embodiments, the loop sequence contains (a) a sequence of (GS)n(SEQ ID NO:23), with n being any integer between 3 and 7, inclusive, or(b) a rationally redesigned sequence via ensemble-based protein design.In some embodiments, the modified gp140 protein is covalently fused tothe nanoparticle platform. In various embodiments, the nanoparticleplatform contains a trimeric sequence. In some of these embodiments, thenanoparticle platform is dihydrolipoyl acyltransferase (E2P), ferritin,or lumazine synthase (LS). In some embodiments, the nanoparticleplatform has one or more 3-fold axes on the surface with the N-terminusof each monomer subunit being in close proximity to the 3-fold axis, andthe spacing of the three N-termini matching the spacing of the C-terminiof the modified gp140 protein trimer. In some embodiments, theC-terminus of the modified gp140 protein sequence is fused to theN-terminus of the subunit of the nanoparticle platform sequence. In someembodiments, the nanoparticle platform contains a self-assemblingnanoparticle with a diameter of about 25 nm or less that is assembledfrom 12 or 24 subunits. Some HIV-1 vaccine compositions of the inventioncan further contain an adjuvant.

In some HIV-1 vaccine compositions of the invention, the gp140 trimer isderived from HIV-1 strain BG505, with a loop sequence as shown in SEQ IDNO:6. Some of the compositions further contains (a) a linker sequence(G₄S)₂ (SEQ ID NO:22) that substitutes for residues 508-511 at thecleavage site, and (b) an engineered disulfide bond between residuesA501C and T605C. In another aspect, the invention provides isolated orrecombinant polynucleotides that encode the HIV-1 fusion immunogens andnanoparticles displaying the immunogens as described herein, as well asexpression vectors and host cells harboring such polynucleotidesequences. In still another aspect, the invention provides methods ofpreventing HIV-1 infection in a subject. These methods entailadministering to the subject a therapeutically effective amount of theHIV-1 immunogen or vaccine composition described herein. Theadministration of the immunogen results in prevention of HIV-1 infectionin the subject. In a related aspect, the invention provides methods oftreating HIV-1 infection or eliciting an immune response against HIV-1in a subject. These methods involve administering to the subject apharmaceutical composition that contains a therapeutically effectiveamount of the HIV-1 immunogen or vaccine composition described herein.

A further understanding of the nature and advantages of the presentinvention may be realized by reference to the remaining portions of thespecification and claims.

DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates computational redesign of HR1 N-terminus and cleavagesite. (a) Atomic model and molecular surface of BG505 SOSIP.664 trimer(PDB ID: 4TVP) with gp120 and two regions of gp41_(ECTO) (residues518-547 and 569-664) within one gp140 protomer. A zoomed-in view of thegp140 structure surrounding the HR1 N-terminus (residues 548-568) andthe cleavage site-containing region (residues 505-518) is shown on theright with the structural gaps connected by black dotted lines. (b)Schematic presentation of the HR1 redesign. (c) Computational procedurefor ensemble-based de novo protein design of the HR1 region (residues548-568). After local backbone sampling in torsion space (step 1) andexhaustive search in sequence-structure space (step 2), the designedsequences #1-5 (SEQ ID NOs:11-15, respectively) are ranked by energy(step 3) prior to manual selection of candidates for experimentalvalidation.

FIG. 2 shows design and validation of a generic HR1 loop sequence(linker), (GS)₄ (SEQ ID NO:24), to stabilize Env trimer. (a) Schematicrepresentation of a generic HR1 linker (HR1-G) design. (b) SEC profilesof SOSIP and HR1-G trimers from a Superdex 200 10/300 column for clade-ABG505 (top, left), clade-B JRFL (top, right), clade-C DU172.17 (middle,two HR1 redesigns obtained from ensemble-based de novo protein designincluded on the right), and B′/C recombinant strain CH115.12 (bottom,CSF-SOS included on the right). The UV value of the trimer peak and theratios of UV values for aggregate peak (at 9 mL) and dimer/monomer peak(at 12 mL) relative to the trimer peak (at 10.5 mL) are labeled.

FIG. 3 shows ensemble-based protein design of the HR1 region with looplengths of 8 and 10 residues. (a) Conformational ensembles of 8- (left)and 10-residue (right) HR1 loops with gp120 and two gp41_(ECTO) regions(518-547 and 569-664) within one gp140 protomer. (b) Cα root-mean-square(RMS) fluctuation of 8- (upper panel) and 10-residue (lower panel)redesigned HR1 loops. (c) Correlation between RAPDF score and Cαroot-mean-square (RMS) fluctuation determined for 8- (left) and10-residue (right) redesigned HR1 loops. (d) 5 top-ranking sequencesmanually selected for 8-residue HR1 redesign (left; SEQ ID NOs:6-10,respectively) and 10-residue HR1 redesign (right; SEQ ID NOs:1-5,respectively) between residues G547 and T569. HR1 sequence encompassingits N-terminus (SEQ ID NO:27), including residues 548-568 (SEQ ID NO:28)being replaced by the loop sequences, is shown above these top rankingloop sequences. The region in WT SOSIP.664 that was subjected tocomputational design.

FIG. 4 shows ensemble-based protein design of the cleavagesite-containing region (500-519) and biochemical characterization of top5 designs. (a) Conformational ensemble of 8-residue loops connectingR500 and F519 (left), Cα RMSF distribution of 8-residue loops (upperright), and correlation between RAPDF score and Cα RMSF (lower right).(b) 5 top-ranking 8-residue CST designs (SEQ ID NOs:16-20, respectively)are shown below the cleavage site-containing sequence (SEQ ID NO:21).The region in WT SOSIP.664 that was subjected to computational design ishighlighted. (c) BN-PAGE of five 293 F-expressed, GNL-purified cleavagesite truncated (CST) BG505 constructs, CST1-5, after SEC on a Superdex200 10/300 column. For each trimer construct, the range of SEC fractionsis labeled. For CST 1, trimer, dimer, monomer, and an unknown Env formare labeled on the BN gel.

DETAILED DESCRIPTION I. Overview

The goal of vaccine development for human immunodeficiency virus type-1(HIV-1) is to induce protective or therapeutic broadly neutralizingantibody (bNAb) responses by vaccination. All bNAbs identified thus fartarget the envelope glycoprotein (Env) trimer on the surface of HIV-1virions. The precursor Env protein, gp160, is trafficked from theendoplasmic reticulum (ER) to the Golgi and cleaved by cellularproteases of the furin family into its mature form. The cleaved Envtrimer engages host receptors to mediate viral entry and is the primarytarget of humoral immune responses. Functional Env is a trimer ofheterodimers, each containing a receptor-binding protein, gp120, and atransmembrane protein, gp41, which are held together by non-covalentinteractions. This mature form of Env is metastable as it is poised toundergo dramatic and irreversible conformational changes upon binding tohost receptor and co-receptor to mediate membrane fusion. Envmetastability also facilitates immune evasion by causing gp120 sheddingand generating a diverse assortment of native, more open and non-nativeconformations.

Various strategies have been proposed in attempts to overcome Envmetastability, and to create stable, homogeneous gp140 trimers forstructural and vaccine studies. For example, development of the BG505SOSIP.664 gp140 trimer (Sanders et al., PLoS Pathog. 9(9):e1003618,2013) has facilitated high-resolution structural analyses, provided arational basis for trimer-based vaccine design, allowed expansion of theSOSIP design to other HIV-1 strains and incorporation of new stabilizingmutations, and removal of furin dependency by cleavage sitemodification. However, a premium is placed on trimer purification inorder to minimize unwanted Env forms and misfolded trimers. Complexmethods such as bNAb affinity purification, negative selection, andmulti-cycle SEC have been developed for trimer purification, which cancertainly be adapted for industrial scale production but will likelyrequire special considerations. It is plausible that trimer impurity andgeneral protein production inefficiency are linked to the fundamentalcauses of metastability that have not been completely solved by previousHIV-1 trimer designs.

The present invention is predicated in part on the present inventors'development of computationally redesigned HIV-1 Env trimer molecules asvaccine immunogens. As detailed in the Examples below, the inventorsinvestigated the primary causes of HIV-1 trimer metastability andexplored alternative trimer designs. The inventors hypothesized that thedisorder observed at the HR1 N-terminus (residues 548-568) is indicativeof metastability that could potentially be minimized by proteinengineering. The inventors redesigned a largely disordered bend inheptad region 1 (HR1) that connects the long, central HR1 helix to thefusion peptide region, substantially improving the yield of well-foldedtrimers. Additionally the cleavage site between gp120 and gp41 wasreplaced with various linkers in the context of the HR1 redesign.Specifically, the inventors tested 10 BG505 trimers with the N-terminusregion of HR1 redesigned computationally. These constructs showedsubstantially higher trimer yield and purity, with SOSIP-like propertiesdemonstrated by crystal structures, EM, and antibody binding. Theinventors then examined the structural and antigenic effect of replacingthe furin cleavage site between gp120 and gp41 with a linker in thecontext of a selected HR1 redesign. These studies uncovered thesensitivity of gp140 folding to modification of this proteolytic site,with a fusion intermediate state observed for trimers with short linkerslacking the SOS mutation. By contrast, the HR1-redesigned trimers with along linker, termed uncleaved pre-fusion-optimized (UFO) trimers,adopted a native-like conformation that resembled many salient featuresof the SOSIP trimer. Additionally, the inventors demonstrated theutility of a generic HR1 linker in trimer stabilization for diversestrains of HIV-1. Further studies undertaken by the inventors showedthat the engineered gp41 domains described herein can be used to pairwith a gp120 polypeptide from many different HIV-1 strains or subtypesto form “chimeric” gp140 trimers, e.g., “UFO-BG” or “UFO-U” asexemplified herein. Together, these studies demonstrated a generalapproach for stabilization of Env trimers from diverse HIV-1 strains.

Other than the gp140-derived soluble trimer immunogens with modified HR1region, the inventors further investigated the display of trimeric HIV-1antigens on nanoparticles with an in-depth structural and antigeniccharacterization. The inventors hypothesized that the trimeric Envantigens, such as V1V2 and gp120, can be presented in native-likeconformations around the threefold axes on the surface of nanoparticles.To test this hypothesis, the inventors designed constructs containingV1V2 and gp120 fused to the N-terminus of ferritin subunit. Thesechimeric antigens assembled into nanoparticles with high affinity forbNAbs targeting the apex as well as other key epitopes consistent withnative-like trimer conformations. The inventors then investigated theparticulate display of a stabilized gp140 trimer with a redesignedheptad repeat 1 (HR1) bend that showed substantial improvement in trimerpurity. To facilitate this analysis, the inventors designed threegp140-ferritin constructs containing different linkers, with gp41truncated at either position 664 or 681. While all gp140-ferritinnanoparticles bound to the apex-directed bNAbs with sub-picomolaraffinities, the MPER-containing gp140 nanoparticle could also berecognized by MPER-specific bNAb 4E10. In addition to ferritin, theinventors also examined the utility of a large, 60-meric E2pnanoparticle to present gp120 and gp140 trimers. As demonstrated herein,the gp140-E2p nanoparticle carrying 20 well-folded trimers demonstratedefficient particle assembly and desired antigenicity.

In accordance with these exemplified studies, the invention providesvarious HIV-1 vaccine immunogens and their clinical applications. SomeHIV-1 vaccine immunogens of the invention are soluble gp140-derivedprotein that harbors a modified N-terminus of the HR1 region in gp41 asdisclosed herein. Some HIV-1 immunogens of the invention contain anHIV-1 Env-derived trimer protein that is presented on a nanoparticleplatform. Therapeutic and preventive uses of the HIV-1 vaccinecompositions of the invention are also provided in the invention.

Unless otherwise specified herein, the vaccine immunogens of theinvention, the encoding polynucleotides, expression vectors and hostcells, as well as the related therapeutic applications, can all begenerated or performed in accordance with the procedures exemplifiedherein or routinely practiced methods well known in the art. See, e.g.,Methods in Enzymology, Volume 289: Solid-Phase Peptide Synthesis, J. N.Abelson, M. I. Simon, G. B. Fields (Editors), Academic Press; 1stedition (1997) (ISBN-13: 978-0121821906); U.S. Pat. Nos. 4,965,343, and5,849,954; Sambrook et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Press, N.Y., (3^(rd) ed., 2000); Brent et al., CurrentProtocols in Molecular Biology, John Wiley & Sons, Inc. (ringbou ed.,2003); Davis et al., Basic Methods in Molecular Biology, ElsevierScience Publishing, Inc., New York, USA (1986); or Methods inEnzymology: Guide to Molecular Cloning Techniques Vol. 152, S. L. Bergerand A. R. Kimmerl Eds., Academic Press Inc., San Diego, USA (1987);Current Protocols in Protein Science (CPPS) (John E. Coligan, et. al.,ed., John Wiley and Sons, Inc.), Current Protocols in Cell Biology(CPCB) (Juan S. Bonifacino et. al. ed., John Wiley and Sons, Inc.), andCulture of Animal Cells: A Manual of Basic Technique by R. Ian Freshney,Publisher: Wiley-Liss; 5th edition (2005), Animal Cell Culture Methods(Methods in Cell Biology, Vol. 57, Jennie P. Mather and David Barneseditors, Academic Press, 1st edition, 1998). The following sectionsprovide additional guidance for practicing the compositions and methodsof the present invention.

II. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which this invention pertains. The following referencesprovide one of skill with a general definition of many of the terms usedin this invention: Academic Press Dictionary of Science and Technology,Morris (Ed.), Academic Press (1^(st) ed., 1992); Oxford Dictionary ofBiochemistry and Molecular Biology, Smith et al. (Eds.), OxfordUniversity Press (revised ed., 2000); Encyclopaedic Dictionary ofChemistry, Kumar (Ed.), Anmol Publications Pvt. Ltd. (2002); Dictionaryof Microbiology and Molecular Biology, Singleton et al. (Eds.), JohnWiley & Sons (3^(rd) ed., 2002); Dictionary of Chemistry, Hunt (Ed.),Routledge (1^(st) ed., 1999); Dictionary of Pharmaceutical Medicine,Nahler (Ed.), Springer-Verlag Telos (1994); Dictionary of OrganicChemistry, Kumar and Anandand (Eds.), Anmol Publications Pvt. Ltd.(2002); and A Dictionary of Biology (Oxford Paperback Reference), Martinand Hine (Eds.), Oxford University Press (4^(th) ed., 2000). Furtherclarifications of some of these terms as they apply specifically to thisinvention are provided herein.

As used herein, the singular forms “a,” “an,” and “the,” refer to boththe singular as well as plural, unless the context clearly indicatesotherwise. For example, “an Env-derived trimer” can refer to both singleor plural Env-derived trimer molecules, and can be considered equivalentto the phrase “at least one Env-derived trimer.”

Unless otherwise noted, the terms “antigen” and “immunogen” are usedinterchangeably to refer to a substance, typically a protein, which iscapable of inducing an immune response in a subject. The terms alsorefer to proteins that are immunologically active in the sense that onceadministered to a subject (either directly or by administering to thesubject a nucleotide sequence or vector that encodes the protein) isable to evoke an immune response of the humoral and/or cellular typedirected against that protein. Thus, in some embodiments, the term“immunogen” can broadly encompass polynucleotides that encodepolypeptide or protein antigens described herein.

Conservative amino acid substitutions providing functionally similaramino acids are well known in the art. The following six groups eachcontain amino acids that are conservative substitutions for oneanother: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid(D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); Arginine (R),Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). Not all residuepositions within a protein will tolerate an otherwise “conservative”substitution. For instance, if an amino acid residue is essential for afunction of the protein, even an otherwise conservative substitution maydisrupt that activity, for example the specific binding of an antibodyto a target epitope may be disrupted by a conservative mutation in thetarget epitope.

Epitope refers to an antigenic determinant. These are particularchemical groups or peptide sequences on a molecule that are antigenic,such that they elicit a specific immune response, for example, anepitope is the region of an antigen to which B and/or T cells respond.Epitopes can be formed both from contiguous amino acids or noncontiguousamino acids juxtaposed by tertiary folding of a protein.

Effective amount of a vaccine or other agent that is sufficient togenerate a desired response, such as reduce or eliminate a sign orsymptom of a condition or disease, such as AIDS. For instance, this canbe the amount necessary to inhibit viral replication or to measurablyalter outward symptoms of the viral infection, such as increase of Tcell counts in the case of an HIV-1 infection. In general, this amountwill be sufficient to measurably inhibit virus (for example, HIV)replication or infectivity. When administered to a subject, a dosagewill generally be used that will achieve target tissue concentrations(for example, in lymphocytes) that has been shown to achieve in vitroinhibition of viral replication. In some examples, an “effective amount”is one that treats (including prophylaxis) one or more symptoms and/orunderlying causes of any of a disorder or disease, for example to treatHIV. In one example, an effective amount is a therapeutically effectiveamount. In one example, an effective amount is an amount that preventsone or more signs or symptoms of a particular disease or condition fromdeveloping, such as one or more signs or symptoms associated with AIDS.

Ferritin is a globular protein found in all animals, bacteria, andplants. It acts primarily to control the rate and location ofpolynuclear Fe(III)₂O₃ formation through the transportation of hydratediron ions and protons to and from a mineralized core. The globular formof ferritin is made up of monomeric subunit proteins (also referred toas monomeric ferritin subunits), which are polypeptides having amolecule weight of approximately 17-20 kDa.

As used herein, a fusion protein is a recombinant protein containingamino acid sequence from at least two unrelated proteins that have beenjoined together, via a peptide bond, to make a single protein. Theunrelated amino acid sequences can be joined directly to each other orthey can be joined using a linker sequence. As used herein, proteins areunrelated, if their amino acid sequences are not normally found joinedtogether via a peptide bond in their natural environment(s) (e.g.,inside a cell). For example, the amino acid sequences of monomericsubunits that make up ferritin, and the amino acid sequences of HIV-1gp120 or gp41 glycoproteins are not normally found joined together via apeptide bond.

HIV-1 envelope protein (Env) is initially synthesized as a longerprecursor protein of 845-870 amino acids in size, designated gp160.gp160 forms a homotrimer and undergoes glycosylation within the Golgiapparatus. In vivo, gp160 glycoprotein is endo-proteolytically processedto the mature envelope glycoproteins gp120 and gp41, which arenoncovalently associated with each other in a complex on the surface ofthe virus. The gp120 surface protein contains the high affinity bindingsite for human CD4, the primary receptor for HIV, as well as domainsthat interact with fusion coreceptors, such as the chemokine receptorsCCR5 and CXCR4. The gp41 protein spans the viral membrane and containsat its amino-terminus a sequence of amino acids important for the fusionof viral and cellular membranes. The native, fusion-competent form ofthe HIV-1 envelope glycoprotein complex is a trimeric structure composedof three gp120 and three gp41 subunits. The receptor-binding (CD4 andco-receptor) sites are located in the gp120 moieties, whereas the fusionpeptides are located in the gp41 components. Exemplary sequence ofwildtype gp160 polypeptides are shown in GenBank, e.g., under accessionnumbers AAB05604 and AAD12142.

gp140 refers to an oligomeric form of HIV envelope protein, whichcontains all of gp120 and the entire gp41 ectodomain.

gp120 is an envelope protein of the Human Immunodeficiency Virus (HIV).gp120 contains most of the external, surface-exposed, domains of the HIVenvelope glycoprotein complex, and it is gp120 which binds both tocellular CD4 receptors and to cellular chemokine receptors (such asCCR5). The mature gp120 wildtype polypeptides have about 500 amino acidsin the primary sequence. Gp120 is heavily N-glycosylated giving rise toan apparent molecular weight of 120 kD. The polypeptide is comprised offive conserved regions (C1-05) and five regions of high variability(V1-V5). In its tertiary structure, the gp120 glycoprotein is comprisedof three major structural domains (the outer domain, the inner domain,and the bridging sheet) plus the variable loops. See, e.g., Wyatt etal., Nature 393, 705-711, 1998; and Kwong et al., Nature 393, 649-59,1998. The inner domain is believed to interact with the gp41 envelopeglycoprotein, while the outer domain is exposed on the assembledenvelope glycoprotein trimer.

Variable region 1 and Variable Region 2 (V1/V2 domain) of gp120 arecomprised of about 50-90 residues which contain two of the most variableportions of HIV-1 (the V1 loop and the V2 loop), and one in ten residuesof the V1/V2 domain are N-glycosylated.

gp41 is a proteolytic product of the precursor HIV envelope protein. Itcontains an N-terminal fusion peptide (FP), a transmembrane domain, aswell as an ectodomain that links the fusion peptide and a transmembranedomain. gp41 remains in a trimeric configuration and interacts withgp120 in a non-covalent manner. The amino acid sequence of an exemplarygp41 is set forth in GenBank, under Accession No. CAD20975.

BG505 SOSIP.664 gp140 is a HIV-1 Env immunogen developed with the gp140trimer from clade-A strain BG505. It contains a covalent linkage betweenthe cleaved gp120 and gp41_(ECTO) with an engineered disulfide bond(termed SOS). In addition, it has an I559P mutation (termed IP) todestabilize the gp41 post-fusion conformation and also a truncation ofthe membrane-proximal external region (MPER) at residue 664 to improvesolubility. This HIV-1 immunogen has an outstanding antigenic profileand excellent structural mimicry of the native spike. Using the SOSIPtrimer as a sorting probe, new bNAbs have been identified andcharacterized. The SOSIP design has also been extended to other HIV-1strains and permitted the incorporation of additional stabilizingmutations. Recently, immunogenicity of SOSIP trimers in rabbits andnonhuman primates was reported, paving the way for human vaccine trials.

HXB2 numbering system is a reference numbering system for HIV proteinand nucleic acid sequences, using HIV-1 HXB2 strain sequences as areference for all other HIV strain sequences. The person of ordinaryskill in the art is familiar with the HXB2 numbering system, and thissystem is set forth in “Numbering Positions in HIV Relative to HXB2CG,”Bette Korber et al., Human Retroviruses and AIDS 1998: A Compilation andAnalysis of Nucleic Acid and Amino Acid Sequences. Korber B, Kuiken C L,Foley B, Hahn B, McCutchan F, Mellors J W, and Sodroski J, Eds.Theoretical Biology and Biophysics Group, Los Alamos NationalLaboratory, Los Alamos, N. Mex.

Immunogenic surface is a surface of a molecule, for example a proteinsuch as gp120, capable of eliciting an immune response. An immunogenicsurface includes the defining features of that surface, for example thethree-dimensional shape and the surface charge. In some examples, animmunogenic surface is defined by the amino acids on the surface of aprotein or peptide that are in contact with an antibody, such as aneutralizing antibody, when the protein and the antibody are boundtogether. A target epitope includes an immunogenic surface. Immunogenicsurface is synonymous with antigenic surface.

Immune response refers to a response of a cell of the immune system,such as a B cell, T cell, or monocyte, to a stimulus. In someembodiment, the response is specific for a particular antigen (an“antigen-specific response”). In some embodiments, an immune response isa T cell response, such as a CD4+ response or a CD8+ response. In someother embodiments, the response is a B cell response, and results in theproduction of specific antibodies.

Immunogenic composition refers to a composition comprising animmunogenic polypeptide that induces a measurable CTL response againstvirus expressing the immunogenic polypeptide, or induces a measurable Bcell response (such as production of antibodies) against the immunogenicpolypeptide.

Sequence identity or similarity between two or more nucleic acidsequences, or two or more amino acid sequences, is expressed in terms ofthe identity or similarity between the sequences. Sequence identity canbe measured in terms of percentage identity; the higher the percentage,the more identical the sequences are. Homologs or orthologs of nucleicacid or amino acid sequences possess a relatively high degree ofsequence identity/similarity when aligned using standard methods.Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smith &Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol.Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp,CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988;Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; andPearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J.Mol. Biol. 215:403-10, 1990, presents a detailed consideration ofsequence alignment methods and homology calculations.

Rotational symmetry, also known in biological contexts as radialsymmetry, refers to the property of an object that looks the same aftera certain amount of rotation. An object may have more than onerotational symmetry; for instance, if reflections or turning it over arenot counted. The degree of rotational symmetry is how many degrees theshape has to be turned to look the same on a different side or vertex.It cannot be the same side or vertex. Rotational symmetry of order n,also called n-fold rotational symmetry, or discrete rotational symmetryof the nth order, with respect to a particular point (in 2D) or axis (in3D; e.g., 3-fold axis described herein) means that rotation by an angleof 360°/n (180°, 120°, 90°, 72°, 60°, 51 3/7°, etc.) does not change theobject.

Bacteriophage Q_(β) (Q_(β) or Q as denoted herein) is an icosahedralvirus with a diameter of 25 nm. Its host is Escherichia coli. Q_(β)enters its host cell through the side of the F pilus. The genome ofQ_(β) is 4217 nucleotides long. The genome has three open reading framesand encodes four proteins: A1, A2, CP and qβ replicase. See, e.g., vanDuin et al., “Single-stranded RNA phages. Chapter 15”. In Calendar, R.L. The Bacteriophages (Second ed., 2006). Oxford University Press. pp.175-196. The genome of Q_(β) is highly structured, which regulates geneexpression and protects the genome from host RNases.

The term “subject” refers to any animal classified as a mammal, e.g.,human and non-human mammals. Examples of non-human animals include dogs,cats, cattle, horses, sheep, pigs, goats, rabbits, and etc. Unlessotherwise noted, the terms “patient” or “subject” are used hereininterchangeably. Preferably, the subject is human.

The term “treating” or “alleviating” includes the administration ofcompounds or agents to a subject to prevent or delay the onset of thesymptoms, complications, or biochemical indicia of a disease (e.g., anHIV infection), alleviating the symptoms or arresting or inhibitingfurther development of the disease, condition, or disorder. Subjects inneed of treatment include those already suffering from the disease ordisorder as well as those being at risk of developing the disorder.Treatment may be prophylactic (to prevent or delay the onset of thedisease, or to prevent the manifestation of clinical or subclinicalsymptoms thereof) or therapeutic suppression or alleviation of symptomsafter the manifestation of the disease.

Vaccine refers to a pharmaceutical composition that elicits aprophylactic or therapeutic immune response in a subject. In some cases,the immune response is a protective immune response. Typically, avaccine elicits an antigen-specific immune response to an antigen of apathogen, for example a viral pathogen, or to a cellular constituentcorrelated with a pathological condition. A vaccine may include apolynucleotide (such as a nucleic acid encoding a disclosed antigen), apeptide or polypeptide (such as a disclosed antigen), a virus, a cell orone or more cellular constituents.

Virus-like particle (VLP) refers to a non-replicating, viral shell,derived from any of several viruses. VLPs are generally composed of oneor more viral proteins, such as, but not limited to, those proteinsreferred to as capsid, coat, shell, surface and/or envelope proteins, orparticle-forming polypeptides derived from these proteins. VLPs can formspontaneously upon recombinant expression of the protein in anappropriate expression system. Methods for producing particular VLPs areknown in the art. The presence of VLPs following recombinant expressionof viral proteins can be detected using conventional techniques known inthe art, such as by electron microscopy, biophysical characterization,and the like. See, for example, Baker et al. (1991) Biophys. J.60:1445-1456; and Hagensee et al. (1994) J. Virol. 68:4503-4505. Forexample, VLPs can be isolated by density gradient centrifugation and/oridentified by characteristic density banding. Alternatively,cryoelectron microscopy can be performed on vitrified aqueous samples ofthe VLP preparation in question, and images recorded under appropriateexposure conditions.

III. Modified HIV-1 gp140 Proteins and Immunogens with Redesigned HR1Region

HIV-1 Env is a heterodimer of a transmembrane glycoprotein (gp41) and asurface glycoprotein (gp120). These dimers are organized as trimers onthe surface of the viral membrane. The HIV-1 trimer immunogens of theinvention are formed of a gp140-related protein that contains agp120-derived polypeptide and a gp41-derived polypeptide with aredesigned N-terminus (residues 548-568) of the heptad region 1 (HR1) ingp41. The gp140-related protein should maintain an appropriate trimericstructure described herein (e.g., a native-like trimeric conformation).The gp120-derived polypeptide and the gp41-derived polypeptide can beassociated non-covalently as in the natural HIV-1 gp140 protein orcovalently linked via a linker sequence described herein. The wellcharacterized gp120 glycoprotein contains the core and several variableloops or domains (e.g., the V1V2 domain and the V3 domain). Variousgp120-derived polypeptides can be employed in the practice of theinvention. The gp120-derived polypeptide does not have to contain thefull-length sequence of a wildtype gp120 glycoprotein. Thus, thegp120-derived polypeptide can be, e.g., the natural gp120 protein, theV1V2 domains of the gp120 glycoprotein, the gp120 core (i.e., the innerdomain and the outer domain), and just the outer domain of gp120 core.In some embodiments, the employed gp120-derived polypeptide encompassesits gp41-interactive region and the antigenic epitopes (e.g., the outerdomain).

Typically, the gp140-derived polypeptide should harbor and expose thenative epitopes (e.g., “sites of HIV-1 vulnerability” or “broadlyneutralizing epitopes”) recognized by one or more of the wellcharacterized HIV bnAbs (e.g., PG9, PG16, CH03, PGDM1400, VRC01, 4E10and 10E8). For example, PG9 is a broadly neutralizing monoclonalantibody that specifically binds to the V1/V2 domain of HIV-1 gp120 andprevents HIV-1 infection of target cells (see, e.g., Walker et al.,Nature, 477:466-470, 2011; and WO/2010/107939). In addition, sequenceswith conservative amino acid substitutions or sequences that aresubstantially identical to the gp140-derived polypeptide exemplifiedherein can also be used in the invention. In various embodiments, thevaccine immunogens of the invention are further characterized by theirantigenicity of specifically binding to one or more (e.g., 2, 3, 4, 5 ormore) of the well known HIV-1 bnAbs (e.g., PG9, PG16, CH03, PGDM1400,VRC01, 4E10 and 10E8). Such antigenicity can be readily assessed viamethods routinely practiced in the art, e.g., the Octet measurement(ForteBio, Inc.). See, e.g., Fera et al., Proc. Natl. Acad. Sci. USA.111: 10275-10280, 2014; and McGuire et al., J. Virol. 88: 2645-2657,2014.

Other than the gp120-derived polypeptide, the gp140-related protein forproducing the HIV-1 trimer immunogens of the invention also contains agp41-derived polypeptide with a redesigned N-terminus (residues 548-568)of the heptad region 1 (HR1). In some embodiments, the gp120 and gp41polypeptides in the engineered gp140 immunogens of the invention arederived from the same HIV-1 strain or subtype. In some embodiments, thegp120 and gp120 polypeptides in the gp140 protein are derived fromdifferent HIV-1 strains or subtypes. For example, as exemplified herein,a modified gp41 from strain BG505 or a universal gp41 domain derivedfrom the HIV-1 sequence database can be combined with gp120 from variousother HIV-strains or subtypes to form different chimeric gp140 trimerimmunogens. The modified gp41-derived polypeptide in the engineeredgp140 immunogens of the invention typically harbors the HR1 region ofthe native gp41 protein excerpt for the N-terminus modificationdescribed herein. The HR1 region undergoes drastic conformational changeduring vial fusion with host cells. Preferably, the gp41-derivedpolypeptide is a soluble polypeptide that has the transmembrane regiontruncated, e.g., a polypeptide containing the ectodomain (gp41_(ECTO))or a polypeptide containing the fusion peptide and the ectodomain. Invarious embodiments, the 21 residue N-terminus of HR1 (residues 548-568)of the gp41-derived polypeptide is replaced with a shorter loop sequenceto stabilize the pre-fusion gp140 structure. The loop sequence cancontain from about 6 to about 14 amino acid residues. Specific loopsequences suitable for the HIV-1 trimer immunogens of the invention canbe obtained by rational design to ensure proper function (e.g.,stabilizing the pre-fusion conformation of gp140). For example, shorterloop sequences replacing the HR1 N-terminus can be designed via theensemble-based de novo protein design method exemplified herein. Asdetailed in the Examples herein, almost all HR1 redesigns based on thecomputational method showed substantial improvement in terms of trimeryield and purity.

In some embodiments, the inserted loop sequence replacing the HR1N-terminus contains 10 amino acid residues. Specific examples of suchloop sequences are shown in SEQ ID NOs:1-5. In some other embodiments,the substituting loop sequence contains 8 amino acid residues. Examplesof such loop sequences are shown in SEQ ID NOs:6-15. In still some otherembodiments, the loop sequence replacing the HR1 N-terminus can containabout 6, 7, 9, 11, 12, 13, or 14 amino acid residues. Such loopsequences can be readily obtained by applying the same rational resignmethods exemplified herein for the 8-residue and 10-residue loopsequences. In some other embodiments, a generic loop sequence containing2-7 tandem repeats of GS ((GS)_(n); SEQ ID NO:23) can be used in theredesign of the HR1 N-terminus. As demonstrated herein (e.g., FIG. 2 a), a generic loop sequence (GS)₄ (SEQ ID NO:24) was shown to beeffective in constructing modified gp140 immunogens from various HIV-1strains.

In addition to the HR1 N-terminus modification, some gp140-derivedproteins for forming HIV-1 trimer immunogen of the invention also havethe protease cleavage site between gp120 and gp41 replaced with a linkersequence to create non-cleavable gp140 protein. As exemplified herein,various cleavage site linkers can be used in the gp140-derived proteinimmunogens of the invention. In various embodiments, the linkers cancontain different amino acid residues of varying length. In someembodiments, the 4-residue cleavage site (i.e., residues 508-511) isreplaced with the linker sequence. For example, the cleavage site can bereplaced with a linker containing one or more tandem repeats of a SGSmotif. Alternatively, the cleavage site can be replaced with a linker of(G₄S)₂ (SEQ ID NO:22). In some other embodiments, a longer cleavagesite-containing region (e.g., residues 501-518) is replaced with thelinker sequence. In some of these embodiments, the linker contains an8-amino acid residue sequence. Some specific linker sequences thatreplace the cleavage site in the gp140-derived protein are shown in SEQID NOs:16-20. As exemplified herein, a combination of the cleavage sitelinker sequence and the redesigned HR1 N-terminus in the gp140immunogens of the invention lead to further improvement in trimer yieldand purity.

In some embodiments, the association between gp120 and gp41 can bestabilized by the introduction of a correctly positioned intermoleculardisulfide bond to make a soluble form of Env, SOS gp140. Such astabilized, native Env complex would increase the time that the trimericgp120-gp41 complex is presented to the immune system. The gp120-gp41interactions in SOS gp140 can also be stabilized by deleting the firstand second variable (V1 and V2) loops and by introducing amino acidsubstitutions into the N-terminal heptad repeat region around position559 of gp41 (see, e.g., WO 03/022869). One such modified gp140 proteinis SOSIP gp140, which contains an I559P substitution. SOSIP gp140 isproperly folded, proteolytically cleaved, substantially trimeric, andhas appropriate receptor binding and antigenic properties. Stability andimmunogenicity of gp140 or other Env-derived trimers can be additionallyenhanced by the trimer-presenting formats described herein.

In some embodiments, the modified gp140-related protein may additionalinclude modified glycan site at residue 332 (T332N). In some otherembodiments, the modified gp140 protein harboring a redesigned HR1N-terminus also has other mutations or alterations introduced at thecleavage site, e.g., replacing REKR (SEQ ID NO:25) with RRRRRR (SEQ IDNO:26). In various embodiments, the C terminus of the modified gp140protein can be truncated to either residue 664 or 681 (according to HXB2nomenclature), resulting in the two gp140 versions like “BG505SOSIP.gp140.664” and “BG505 SOSIP.gp140.681” which are known in the art.Also, the HIV-1 immunogens of the invention can employ the differentgp140 derived proteins from various HIV-1 clades or strains (e.g.,strains BG505 (clade A), JRFL (clade B) CAP45 (clade C), ZM109 (cladeC), DU172.17 (clade C), and CH115.12 (clade B′/C) exemplified herein).HIV-I can be classified into four groups: the “major” group M, the“outlier” group 0, group N, and group P. Within group M, there areseveral genetically distinct clades (or subtypes) of HIV-I. The gp140trimers for the present invention can be derived from any subtype ofHIV, such as groups M, N, O, or P or clade A, B, C, D, F, G, H, J or Kand the like. Sequences encoding HIV-1 Env glycoproteins and methods forthe manipulation and insertion of such nucleic acid sequences intovectors, are known (see, e.g., HIV Sequence Compendium, Division ofAIDS, National Institute of Allergy and Infectious Diseases (2003); HIVSequence Database (hiv-web.lanl.gov/content/hiv-db/mainpage.html);Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Press, N.Y., (3^(rd) ed., 2000); and Brent et al., CurrentProtocols in Molecular Biology, John Wiley & Sons, Inc. (ringbou ed.,2003). Further, there is an HR1-type region in most enveloped virusesthat employ a similar type-1 fusion mechanism, such as influenza virus,Ebola, and respiratory syncytial virus (RSV). The strategy forgenerating HIV-1 gp140 immunogens of the invention can also be employedfor stabilizing Env spikes in designing and producing vaccine immunogensfor the other enveloped viruses.

As detailed below, the gp140-derived protein may be conjugated to thepresenting platform (e.g., nanoparticles or VLPs) via various means.Preferably, the conjugation is achieved via covalent linkage, e.g.,protein fusions or insertions. In some preferred embodiments, theprotein sequence is fused with the presenting platform sequence via alinker sequence. In the various immunogens of the invention, othermodifications can also be made to the gp140-derived trimers or theconjugating partner in order to improve stability or antigenicity.

The various gp140-derived proteins used in the invention can be obtainedor generated in accordance with the protocols exemplified herein ormethods well known in the art. Upon recombinant expression (e.g., inHEK293 F cells as detailed herein), the proteins can be purified by anyof the routinely practiced procedures. See for example Guide to ProteinPurification, ed. Deutscher, Meth. Enzymol. 185, Academic Press, SanDiego, 1990; and Scopes, Protein Purification: Principles and Practice,Springer Verlag, New York, 1982. Substantial purification denotespurification from other proteins or cellular components. A substantiallypurified protein is at least 60%, 70%, 80%, 90%, 95% or 98% pure. Oncepurified, antigenicity and other properties of gp140 trimer immunogensformed of the gp140 derived protein can also be readily examined withstandard methods, e.g., antigenic profiling using known bNAbs andnon-Nabs, differential scanning calorimetry (DSC), electron microscopy,binding analysis via ELISA and Biolayer Light Interferometry (BLI), andco-crystallography analysis as exemplified herein.

IV. Scaffolded HIV-1 Trimer Immunogen Compositions

Other than soluble gp140-based trimer immunogens described above, theinvention also provides HIV-1 immunogens that contain a heterologousscaffold that presents or incorporates a trimeric Env-derived protein.In some embodiments, the heterologous scaffold is a nanoparticle orvirus-like particle (VLP). Various nanoparticle platforms can beemployed in generating the vaccine compositions of the invention. Ingeneral, the nanoparticles employed in the invention need to be formedby multiple copies of a single subunit. Additionally or alternatively,the amino-terminus of the particle subunit has to be exposed and inclose proximity to the 3-fold axis, and the spacing of threeamino-termini has to closely match the spacing of the carboxyol-terminiof various HIV-1 trimeric components. In some preferred embodiments, theimmunogens comprise self-assembling naoparticles with a diameter ofabout 30 nm or less, about 25 nm or less, or about 20 nm or less(usually assembled from 12, 24, or 60 sububits). Such nanoparticlesprovide suitable particle platforms to produce multivalent HIV-1 trimervaccines. When VLP platform is used, the diameter of the VLPs can be asbig as 30 nm, 40 nm, 60 nm or even bigger.

In some embodiments, the HIV-1 trimer-presenting nanoparticles arenaturally existing nanoparticles such as ferritin ion cages with 3-foldaxes on the surface. They allow presentation of multiple copies of thetrimeric component of HIV-1 envelope complex (Env), enabling a series ofmultivalent trimer vaccine candidates. As an example, one of suchnanoparticles is the ferritin nanoparticle from Helicobacter pylori.Ferritin is a globular protein found in all animals, bacteria, andplants. Its primary function is to control the rate and location ofpolynuclear Fe(III)₂O₃ formation through the transportation of hydratediron ions and protons to and from a mineralized core. The globular formof ferritin is made up of monomeric subunit proteins (also referred toas monomeric ferritin subunits), which are polypeptides having amolecule weight of approximately 17-20 kDa.

A monomeric ferritin subunit used in the invention is a full length,single polypeptide of a ferritin protein, or any portion thereof, whichis capable of directing self-assembly of monomeric ferritin subunitsinto the globular form of the protein. Amino acid sequences frommonomeric ferritin subunits of any known ferritin protein can be used toproduce fusion proteins of the present invention, so long as themonomeric ferritin subunit is capable of self-assembling into ananoparticle displaying HIV-1 epitopes on its surface. In addition toferritin, the invention can also employ many other self-assemblingnanoparticles with similar molecular traits. These include, e.g.,molecules with the following PDB IDs: 1JIG (12-mer Dlp-2 from Bacillusanthracis), 1UVH (12-mer DPS from Mycrobacterium smegmatis), 2YGD(24-mer eye lens chaperone αB-crystallin), 3CS0 (24-mer DegP24), 3MH6and 3MH7 (24-mer HtrA proteases), 3PV2 (12-mer HtrA homolog DegQ WT),4A8C (12-mer DegQ from E. coli.), 4A9G (24-mer DegQ from E. coli.), 4EVE(12-mer HP-NAP from Helicobacter pylori strain YS29), and 4GQU (24-merHisB from Mycobacterium tuberculosis).

In some embodiments, the HIV-1 trimer immunogen presenting nanoparticlesare thermostable 60-meric nanoparticles, e.g., dihydrolipoylacyltransferase (E2p) from Bacillus stearothermophilus. In someembodiments, the employed nanoparticles can be lumazine synthase (LS)from Aquifex aeolicus. E2p is a hollow dodecahedron with a diameter of23.2 nm and 12 large openings separating the threefold vertices on theparticle surface. LS, with a diameter of 14.8 nm, is an assembly of 60subunits arranged in a capsid with T ¼ 1 icosahedral symmetry. Asexemplified herein, trimer immunogens presented on these nanoparticles(e.g., E2p) have excellent structural and functional properties,including an optimal size for direct uptake by DCs and increasedrecognition by bNAbs.

Any Env-derived HIV-1 trimer proteins can be used in thenanoparticle-presented vaccine compositions. In some embodiments, thenanoparticles present a native trimeric form of HIV-1 Env basedglycoproteins or domains, e.g., gp140, gp120 or V1V2 domains asexemplified herein (see, e.g., Table 2). In some embodiments, thenanoparticles present a modified gp140 trimer immunogen, e.g., aHR1-modified gp140 trimer described herein. As the receptor-bindingprotein of HIV-1 Env, gp120 has been extensively studied as a vaccineimmunogen, but is now considered suboptimal due to the exposure of anon-neutralizing face that is buried within the native spike. Asdemonstrated herein, display of full-length gp120 with ferritin and E2pnanoparticles can restore the native-like trimer conformation in theabsence of gp41. With SOSIP-like antigenicity and variations in particlesize and surface spacing, these nanoparticles provide versatileplatforms to investigate gp120-based HIV-1 vaccines.

In addition, the Env-derived trimer protein can be obtained from variousHIV-1 strains. In some embodiments, the Env-derived trimer is from HIV-1strain BG505. As exemplifications, V1V2-ferritin nanoparticles wereproduced with trimer proteins of HIV-1 strains ZM109 and CAP45. Alsoexemplified herein are nanoparticles (E2p or ferritin) displaying gp140trimers, full length gp120, full length gp120 with an additionaldisulfide bond to stabilize the gp120 termini, and gp120 molecules ofdifferent lengths. These emplifications indicate that the generalnanoparticle structure and design described herein strategy can beapplied to create multivalent HIV-1 vaccine candidates based on otherHIV-1 strains.

In various embodiments, nanparticle displaying any of these HIV-1Env-derived immunogens can be constructed by fusing the trimer immunogento the subunit of the nanoparticle (e.g., E2p or ferritin subunit). Theantigeniciy and structural integrity of these nanoparticle based HIV-1immunogens can be readily analyzed via standard assays, e.g., antibodybinding assays and negative-stain electron microscopy (EM). Asexemplified herein, the various fusion molecules can all self-assembleinto nanoparticles that display immunogenic epitopes of the Env-derivedtrimer (e.g., gp140). By eliciting a robust trimer-specific bnAbs, thesenanoparticles are useful for vaccinating individuals against a broadrange of HIV-1 viruses.

In some embodiments, the heterologous scaffold that presents orincorporates a trimeric Env-derived protein, e.g., a gp140-derivedtrimer protein described herein, is a virus-like particle (VLP) such asbacteriophage Q_(β) VLP as exemplified herein, or a self-assemblingnanoparticle possessing the same molecular and geometric traits as aVLP. In general, the VLPs to be used in the present invention need tomeet at least one, and preferably all, of the following criteria: (1)the VLP has to be formed by multiple copies of a single subunit; (2) theVLP has to have 3-fold axes displayed on the surface; and (3) theN-terminus of each VLP subunit has to be exposed and in close proximityto the 3-fold axis, and the spacing of three N-termini match the spacingof the C-termini of an HIV-1 trimeric antigen so that the HIV-1 antigencan be fused to the N-terminus of the VLP subunit. Or alternatively, the3-fold axis is surrounded by three surface loops, each from a VLPsubunit, where the HIV-1 antigen can be inserted into the subunit chain.

In various embodiments, the VLP based HIV-1 immunogens of the inventioncan have a minimum of 20-25 epitopes spaced by 5-10 nm, which issufficient for B-cell activation. In some embodiments, the VLPs have adiameter of 30-40 nm and 3-fold axes on the surface, which provide anideal platform to develop multivalent HIV-1 trimer vaccines. In someembodiments, the VLP based HIV-1 immunogens can employs any of the VLPsidentified by the inventors via bioinformatic analysis of an annotateddatabase for icosahedral virus capsids, VIPERdb. These includebacteriophage Q with a 3.5 Å crystal structure (PDB ID: 1QBE), flockhouse virus (FHV) capsid with a 3.5 Å crystal structure (PDB ID: 4FSJ),Orsay virus capsid with a 3.25 Å crystal structure (PDB ID: 4NWV) in thePDB database, and B-cell activating factor (BAFF) with a 3.0 Å crystalstructure (PDB ID: 1JH5), which forms a 60-mer VLP-like assembly. Insome preferred embodiments, bacteriophage Q is used due to its optimalstructural features. Additional VLPs suitable for the invention can bereadily identified via bioinformatic search of similar particle assemblyand subunit structure as that identified for any of these exemplifiedVLPs. For example, bacteriophages MS2 (PDB ID: 2WBH) and P22 (2XYY and2XYZ) have been used to engineer antigen-presenting VLP vaccineplatforms. These two bacteriophage VLPs can also be used to constructmultivalent HIV-1 vaccine immunogens of the invention.

The multivalently scaffolded HIV-1 trimer immunogens of the inventioncan be constructed in accordance with the methods described herein(e.g., Examples 9-13). Various nanoparticle presenting HIV-1 trimerimmunogens are exemplified herein. These include V1V2 trimers presentedon ferritin (SEQ ID NOs:29-31), gp120 trimers presented on ferritin (SEQID NOs:32-34), gp120 trimers presented by E2p or LS (SEQ ID NOs:35-36),gp140 trimers presented on ferritin nanoparticles (SEQ ID NOs:37-39),and gp140 trimer immunogens presented on LS or E2p nanoparticles (SEQ IDNOs:40-41). In general, to construct the VLP presenting HIV-1 trimerimmunogens, the trimer sequences can either be fused with the VLPsequence (e.g., at the N-terminus of the VLP) or inserted into the VLPsequence. In some embodiments, the VLP is fused at its N-terminus withthe HIV-1 Env-derived trimer, e.g., HIV-1 V1V2, gp120, and the twoversions of SOSIP gp140 trimer noted above can be presented on the VLP.In some other embodiments, the HIV-1 Env-derived trimer is inserted intothe VLP. In these embodiments, the HIV-1 trimer can be the V1V2 domainsor the gp120 protein. Since the N- and C-termini of gp140 are distant,this Env-derived trimer is not suited for insertion into the VLP. Asexemplified herein, a series of VLP constructs were generated by fusingHIV-1 V1V2, gp120, and two versions of SOSIP gp140 to the Q subunit, byinserting V1V2 into the surface loops of FHV and Orsay subunits, and byinserting V1V2 and gp120 into a surface loop of BAFF. As detailed in theExamples below, antigenicity and VLP assembly were validated for allQ-based VLPs with antibody binding assays and negative stain electronmicroscopy (EM). Antigenicity was also validated for the FHV-, Orsay-,and BAFF-based VLPs.

V. Vectors and Host Cells for Expressing HIV-1 Immunogens orNanoparticles

The invention provides polynucleotide sequences that encode the HIV-1immunogens or nanoparticles displaying the immunogens as describedherein, expression vectors that harbor the polynucleotide sequences, aswell as host cells that harbor the polynucleotides or expressionconstructs. The cell can be, for example, a eukaryotic cell, or aprokaryotic cell, such as an animal cell, a plant cell, a bacterium, ora yeast. A variety of expression vector/host systems are suitable forexpressing the fusion polypeptides of the invention. Examples include,e.g., microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransfected with virus expression vector (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or transformed with bacterialexpression vectors (e.g., Ti or pBR322 plasmid); or animal cell systems.

Vectors useful for the invention preferably contain sequences operablylinked to the fusion polypeptide coding sequences that permit thetranscription and translation of the encoding polynucleotide sequences.Sequences that permit the transcription of the linked fusion polypeptideencoding sequences include a promoter and optionally also include anenhancer element or elements permitting the strong expression of thelinked sequences. The term “transcriptional regulatory sequences” refersto the combination of a promoter and any additional sequences conferringdesired expression characteristics (e.g., high level expression,inducible expression, tissue- or cell-type-specific expression) on anoperably linked nucleic acid sequence. The promoter sequence can beconstitutive or inducible. Examples of constitutive viral promotersinclude the HSV, TK, RSV, SV40 and CMV promoters. Examples of suitableinducible promoters include promoters from genes such as cytochrome P450genes, heat shock protein genes, metallothionein genes,hormone-inducible genes, such as the estrogen gene promoter, and thelike.

In addition to promoter/enhancer elements, expression vectors of theinvention may further comprise a suitable terminator. Such terminatorsinclude, for example, the human growth hormone terminator, or, for yeastor fungal hosts, the TPI1 (Alber & Kawasaki, J Mol Appl Genet. 1:419-34,1982) or ADH3 terminator (McKnight et al., 1985, EMBO J. 4: 2093-2099).Vectors useful for the invention may also comprise polyadenylationsequences (e.g., the SV40 or Ad5E1b poly(A) sequence), and translationalenhancer sequences (e.g., those from Adenovirus VA RNAs). Further, avector useful for the invention may encode a signal sequence directingthe fusion polypeptide to a particular cellular compartment or,alternatively, may encode a signal directing secretion of the fusionpolypeptide.

In some preferred embodiments, vectors expressing the immunogens andnanoparticles of the invention are viral vectors for mammalianexpression. In general, any viral vector that permits the introductionand expression of sequences encoding the fusion HIV-immunogens of theinvention is acceptable for the invention. Examples of mammalianexpression vectors include the adenoviral vectors, the pSV and the pCMVseries of plasmid vectors, vaccinia and retroviral vectors, as well asbaculovirus. As exemplified herein, the HIV-1 immunogens andnanoparticles of the invention can be expressed from viral vectorphCMV3.

Depending on the specific vector used for expressing the fusionpolypeptide, various known cells or cell lines can be employed in thepractice of the invention. The host cell can be any cell into whichrecombinant vectors carrying a fusion HIV-immunogen of the invention maybe introduced and wherein the vectors are permitted to drive theexpression of the fusion polypeptide is useful for the invention. It maybe prokaryotic, such as any of a number of bacterial strains, or may beeukaryotic, such as yeast or other fungal cells, insect or amphibiancells, or mammalian cells including, for example, rodent, simian orhuman cells. Cells expressing the fusion polypeptides of the inventionmay be primary cultured cells, for example, primary human fibroblasts orkeratinocytes, or may be an established cell line, such as NIH3T3,HEK293, HEK293T HeLa, MDCK, WI38, or CHO cells. In some embodiments, thehost cells for expressing the HIV-1 immunogens or nanoparticles of theinvention can be HEK293F or HEK293S cells as exemplified herein. In someother embodiments, the HIV-1 immunogens or nanoparticles of theinvention can be expressed transiently in ExpiCHO cells. The skilledartisans can readily establish and maintain a chosen host cell type inculture that expresses the fusion immunogene. Many other specificexamples of suitable cell lines that can be used in expressing thefusion polypeptides are described in the art. See, e.g., Smith et al.,1983., J. Virol 46:584; Engelhard, et al., 1994, Proc Nat Acad Sci91:3224; Logan and Shenk, 1984, Proc Natl Acad Sci, 81:3655; Scharf, etal., 1994, Results Probl Cell Differ, 20:125; Bittner et al., 1987,Methods in Enzymol, 153:516; Van Heeke & Schuster, 1989, J Biol Chem264:5503; Grant et al., 1987, Methods in Enzymology 153:516; Brisson etal., 1984, Nature 310:511; Takamatsu et al., 1987, EMBO J 6:307; Coruzziet al., 1984, EMBO J 3:1671; Broglie et al., 1984, Science, 224:838;Winter J and Sinibaldi R M, 1991, Results Probl Cell Differ., 17:85;Hobbs S or Murry L E in McGraw Hill Yearbook of Science and Technology(1992) McGraw Hill New York N.Y., pp 191-196 or Weissbach and Weissbach(1988) Methods for Plant Molecular Biology, Academic Press, New York, pp421-463.

The fusion polypeptide-expressing vectors may be introduced to selectedhost cells by any of a number of suitable methods known to those skilledin the art. For the introduction of fusion polypeptide-encoding vectorsto mammalian cells, the method used will depend upon the form of thevector. For plasmid vectors, DNA encoding the fusion polypeptidesequences may be introduced by any of a number of transfection methods,including, for example, lipid-mediated transfection (“lipofection”),DEAE-dextran-mediated transfection, electroporation or calcium phosphateprecipitation. These methods are detailed, for example, in Brent et al.,supra. Lipofection reagents and methods suitable for transienttransfection of a wide variety of transformed and non-transformed orprimary cells are widely available, making lipofection an attractivemethod of introducing constructs to eukaryotic, and particularlymammalian cells in culture. For example, LipofectAMINE™ (LifeTechnologies) or LipoTaxi™ (Stratagene) kits are available. Othercompanies offering reagents and methods for lipofection include Bio-RadLaboratories, CLONTECH, Glen Research, InVitrogen, JBL Scientific, MBIFermentas, PanVera, Promega, Quantum Biotechnologies, Sigma-Aldrich, andWako Chemicals USA.

For long-term, high-yield production of recombinant fusion polypeptides,stable expression is preferred. Rather than using expression vectorswhich contain viral origins of replication, host cells can betransformed with the fusion polypeptide-encoding sequences controlled byappropriate expression control elements (e.g., promoter, enhancer,sequences, transcription terminators, polyadenylation sites, etc.), andselectable markers. The selectable marker in the recombinant vectorconfers resistance to the selection and allows cells to stably integratethe vector into their chromosomes. Commonly used selectable markersinclude neo, which confers resistance to the aminoglycoside G-418(Colberre-Garapin, et al., J. Mol. Biol., 150:1, 1981); and hygro, whichconfers resistance to hygromycin (Santerre, et al., Gene, 30: 147,1984). Through appropriate selections, the transfected cells can containintegrated copies of the fusion polypeptide encoding sequence.

VI. Pharmaceutical Compositions and Therapeutic Applications

The invention provides pharmaceutical compositions and related methodsof using the HIV-1 immunogens (e.g., soluble gp140-derived proteins ornanoparticles displaying an Env-derived trimer, as well aspolynucleotides encoding the proteins or nanoparticles) described hereinfor preventing and treating HIV-1 infections. In some embodiments, theimmunogens disclosed herein are included in a pharmaceuticalcomposition. The pharmaceutical composition can be either a therapeuticformulation or a prophylactic formulation. Typically, the compositionadditionally includes one or more pharmaceutically acceptable vehiclesand, optionally, other therapeutic ingredients (for example, antibioticsor antiviral drugs). Various pharmaceutically acceptable additives canalso be used in the compositions.

Some of the pharmaceutical compositions of the invention are vaccines.For vaccine compositions, appropriate adjuvants can be additionallyincluded. Examples of suitable adjuvants include, e.g., aluminumhydroxide, lecithin, Freund's adjuvant, MPL™ and IL-12. In someembodiments, the HIV-1 immunogens disclosed herein can be formulated asa controlled-release or time-release formulation. This can be achievedin a composition that contains a slow release polymer or via amicroencapsulated delivery system or bioadhesive gel. The variouspharmaceutical compositions can be prepared in accordance with standardprocedures well known in the art. See, e.g., Remington's PharmaceuticalSciences, 19.sup.th Ed., Mack Publishing Company, Easton, Pa., 1995;Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson,ed., Marcel Dekker, Inc., New York, 1978); U.S. Pat. Nos. 4,652,441 and4,917,893; 4,677,191 and 4,728,721; and 4,675,189.

The pharmaceutical compositions of the invention can be readily employedin a variety of therapeutic or prophylactic applications for treatingHIV-1 infection or eliciting an immune response to HIV-1 in a subject.For example, the composition can be administered to a subject to inducean immune response to HIV-1, e.g., to induce production of broadlyneutralizing antibodies to HIV-1. For subjects at risk of developing anHIV infection, a vaccine composition of the invention can beadministered to provide prophylactic protection against viral infection.Depending on the specific subject and conditions, the pharmaceuticalcompositions of the invention can be administered to subjects by avariety of administration modes known to the person of ordinary skill inthe art, for example, intramuscular, subcutaneous, intravenous,intra-arterial, intra-articular, intraperitoneal, or parenteral routes.In general, the pharmaceutical composition is administered to a subjectin need of such treatment for a time and under conditions sufficient toprevent, inhibit, and/or ameliorate a selected disease or condition orone or more symptom(s) thereof. The immunogenic composition isadministered in an amount sufficient to induce an immune responseagainst HIV-1. For therapeutic applications, the compositions shouldcontain a therapeutically effective amount of the HIV-1 immunogendescribed herein. For prophylactic applications, the compositions shouldcontain a prophylactically effective amount of the HIV-1 immunogendescribed herein. The appropriate amount of the immunogen can bedetermined based on the specific disease or condition to be treated orprevented, severity, age of the subject, and other personal attributesof the specific subject (e.g., the general state of the subject's healthand the robustness of the subject's immune system). Determination ofeffective dosages is additionally guided with animal model studiesfollowed up by human clinical trials and is guided by administrationprotocols that significantly reduce the occurrence or severity oftargeted disease symptoms or conditions in the subject.

For prophylactic applications, the immunogenic composition is providedin advance of any symptom, for example in advance of infection. Theprophylactic administration of the immunogenic compositions serves toprevent or ameliorate any subsequent infection. Thus, in someembodiments, a subject to be treated is one who has, or is at risk fordeveloping, an HIV infection, for example because of exposure or thepossibility of exposure to HIV. Following administration of atherapeutically effective amount of the disclosed therapeuticcompositions, the subject can be monitored for HIV-1 infection, symptomsassociated with HIV-1 infection, or both.

For therapeutic applications, the immunogenic composition is provided ator after the onset of a symptom of disease or infection, for exampleafter development of a symptom of HIV-1 infection, or after diagnosis ofHIV-1 infection. The immunogenic composition can thus be provided priorto the anticipated exposure to HIV virus so as to attenuate theanticipated severity, duration or extent of an infection and/orassociated disease symptoms, after exposure or suspected exposure to thevirus, or after the actual initiation of an infection.

The pharmaceutical composition of the invention can be combined withother agents known in the art for treating or preventing HIV infections.These include, e.g., antibodies or other antiviral agents such asnucleoside reverse transcriptase inhibitors, such as abacavir, AZT,didanosine, emtricitabine, lamivudine, stavudine, tenofovir,zalcitabine, zidovudine, and the like, non-nucleoside reversetranscriptase inhibitors, such as delavirdine, efavirenz, nevirapine,protease inhibitors such as amprenavir, atazanavir, indinavir,lopinavir, nelfinavir, osamprenavir, ritonavir, saquinavir, tipranavir,and the like, and fusion protein inhibitors such as enfuvirtide and thelike. Administration of the pharmaceutical composition and the knownanti-HIV agents can be either concurrently or sequentially.

The HIV-1 vaccine immunogens or pharmaceutical compositions of theinvention can be provided as components of a kit. Optionally, such a kitincludes additional components including packaging, instructions andvarious other reagents, such as buffers, substrates, antibodies orligands, such as control antibodies or ligands, and detection reagents.An optional instruction sheet can be additionally provided in the kits.

EXAMPLES

The following examples are offered to illustrate, but not to limit thepresent invention.

Example 1 Ensemble-Based Protein Design for the HR1 Region

We hypothesized that the N-terminus of HR1 (residues 548-568) is acritical determinant of HIV-1 trimer metastability because it is poisedto elongate during fusion and is disordered in all but one reportedstructure of the SOSIP trimer, where it still appears less orderedcompared to the surrounding regions (FIG. 1 a ). Disorder at the top ofthe long HR1 central helix is somewhat unexpected because this region isat the core of the Env complex; however, this region is expected torefold and become helical in the post-fusion form, as in the equivalentregion of influenza hemagglutinin and other type 1 viral fusion proteins(Wilson et al., Nature 289, 1981), and therefore less ordered in thepre-fusion form or at least adopt a completely different conformation.In the SOSIP design, in addition to an engineered disulfide bond(A501C/T605C), the I559P mutation was introduced to destabilize thepost-fusion state and was critical for production of high-quality Envprotein, strongly supporting the notion that this HR1 region might berelated to Env metastability.

In this study, the HR1 bend was subjected to rational redesign aimed tostabilize the pre-fusion conformation, rather than to just destabilizethe post-fusion conformation as in the BG505 SOSIP.664 trimer (FIG. 1 b). Although this wild-type (WT) HR1 region consists of 21 residues, theCa distance between G547 and T569 is merely 24.8 Å, which is equivalentto a fully extended polypeptide backbone of only 6.3 residues. Here, wedecided to examine two loop lengths—8 and 10 amino acids—for the HR1redesign, allowing for a small degree of flexibility while dramaticallyshortening the WT HR1 loop. We utilized ensemble-based protein design(see Methods) to identify sequences that may stabilize the pre-fusiontrimer structure (FIG. 1 c ). Given a specified loop length, a largeensemble of backbone conformations was generated to bridge the gapbetween G547 and T569 (FIG. 3 a ). For 8-residue loops, the Caroot-mean-square fluctuation (RMSF) ranges from 1.3 to 5.7 Å with anaverage of 2.3 Å, whereas for 10-residue loops, a greater conformationalspace was sampled with an average Cα RMSF of 3.6 Å (FIG. 3 b ). After anexhaustive sampling in sequence space, all designs were ranked by theirenemy scores (FIG. 3 c ). The 5 top-ranking sequences for each looplength, totaling 10, were advanced to experimental validation (FIG. 3 d).

Example 2 Biochemical and Biophysical Characterization of HR1 Redesigns

As demonstrated for SOSIP, sc-gp140, and NFL trimers, biochemical andbiophysical properties provide an initial assessment of trimer designs.Following a similar strategy, we assessed the 10 HR1-redesigned BG505trimers containing the same T332N (to restore the N332 epitope), SOS(A501C/T605C), and R6 mutations as the SOSIP.664 trimer (except forI559P). As noted, various purification protocols can produce trimers ofvarying quality. Here, we adopted a rather simple protocol utilizingmaterials that are readily available to most researchers and can bescaled up in an industrial setting. All constructs were expressedtransiently in HEK293 F cells with co-transfected furin as previouslydescribed (Sanders et al. PLoS Pathog. 9, e1003618, 2013). The secretedEnv proteins were purified using a Galanthus nivalis lectin (GNL) columnfollowed by a single SEC on a Superdex 200 10/300 column. One-literexpression produced sufficient quantities (3-7 mg) of HR1-redesignedtrimers, compared to three separate two-liter expressions for the SOSIPtrimer. Although GNL purification does not yield the purest trimers, itenables the comparison of basic properties for various trimer constructssuch as monomer/dimer and higher multimeric species that would otherwisebe filtered out by more sophisticated purification methods.

We compared the SEC profiles based on simple metrics utilizing theultraviolet 280 nm absorbance values (UV). The UV value of the trimerpeak was used as an indicator of the trimer yield, with the aggregateand dimer/monomer peaks measured as ratios of their UV values versusthat of the trimer peak. The two-liter SOSIP expression showed anaverage UV value of 371 for the trimer peak, with average ratios of 31%and 49% for the aggregate and dimer/monomer peaks, respectively. Thefive 8-residue HR1 redesigns (named HR1-redesign 1-5, respectively)showed significantly increased trimer yield with reduced aggregate anddimer/monomer peaks in the SEC profiles. Overall, HR1 redesigns 1 and 2appeared to be the best performers in this group. For example, the HR1redesign 2 showed a near two-fold increase in the UV value of the trimerpeak, with a 16% and 22% reduction in UV values for aggregate anddimer/monomer peaks relative to SOSIP, indicative of improvement in bothtrimer yield and purity. The five 10-residue HR1 redesigns (namedHR1-redesign 6-10, respectively) presented a similar trend, but lesspronounced improvement. Notwithstanding, HR1 redesign 10 showed a UVvalue for the trimer peak that is comparable to the SOSIP trimer fromtwo-liter expression, with the same low level of unwanted Env species asHR1 redesigns 1 and 2. This finding was consistent with the blue nativepolyacrylamide gel electrophoresis (BN-PAGE) analysis that showed moreconcentrated trimer bands on the gel. The trimer-containing fractionswere eluted at 10.25-10.75 mL for the initial assessment of thermalstability by differential scanning calorimetry (DSC). For all 10 testedHR1 redesigns, the DSC profiles showed similar unfolding peaks with athermal denaturation midpoint (T_(m)) ranging from 65.7 to 69.2° C.,closely resembling the T_(m) of 68.1° C. reported for the SOSIP trimer.

Overall, shortening and redesign of this HR1 region exerted a positiveeffect on the composition of produced Env proteins. In addition toincreasing the trimer yield and reducing other Env species, HR1redesigns retained the thermal stability of parent SOSIP.664 trimer,supporting the notion that this HR1 connecting loop region is a keydeterminant of HIV-1 trimer metastability with respect to expression andthe presence of unwanted Env species.

Example 3 Crystallographic Analysis of Two Representative HR1 Redesigns

HR1 redesigns 1 and 9 were selected for crystallographic analysis. Thesetwo constructs differed not only in the redesigned loop length (8 versus10 amino acids), but also notably in their SEC profiles, with redesign 9displaying higher quantities of dimer, providing an opportunity toexamine how HR1 truncation and design variation affect gp140 trimerstructure. Due to the stringent requirement of sample homogeneity forcrystallization, we prepared the HR1-redesigned and WT SOSIP trimers aspreviously described in Kong et al. Acta Crystallogr. Sect. D-Biol.Crystallogr. 71, 2099-2108, 2015. In brief, all trimers were produced inN-acetylglucosaminyltransferase I-negative (GnTI^(−/−)) HEK293 S cellsand purified using a 2G12 affinity column followed by SEC on a Superdex200 16/600 column. For the WT SOSIP trimer, the SEC profile displayed anotable aggregate peak of high molecular weight, with a UV value that is58% of the trimer peak and a lower peak containing monomeric gp140. Bycontrast, the 2G12-purified HR1 redesigns showed a marked improvement intrimer yield and purity. Of particular note, the HR1 redesign 1 showedan almost undetectable level of gp140 monomer, whereas HR1 redesign 9still contained a small fraction of monomer. Nevertheless, the SECprofiles of 293 S-expressed, 2G12-purified trimers are consistent withthat of the 293 F-expressed, GNL-purified trimers described above. Thisfinding was further confirmed by BN-PAGE and the thermal stability ofthe modified trimers measured by DSC, suggesting that the improvedtrimer properties are an intrinsic feature of the HR1 redesigns andindependent of the expression and purification systems.

Co-crystallization with antigen-binding fragments (Fabs) of PGT128 and8ANC195 yielded complex structures at resolutions of 6.9 and 6.3 Å forthe 8- and 10-residue HR1-redesigned trimers, respectively (Table 1).Overall, the redesigned trimers displayed nearly identical structures tothat of the SOSIP trimer at this modest solution, with Caroot-mean-square deviations (RMSD) ≤0.25 Å. Thus the results confirmedthat gp140 trimers with shortened and redesigned HR1 still adopt aSOSIP-like pre-fusion structure. Limited by the resolution, we couldonly determine the approximate backbone conformation of the redesignedHR1 loop, which alluded to how these two distinct designs stabilize thepre-fusion trimer. We speculated that the shortened loop length (8 or 10versus 21 amino acids) and a redesigned sequence disrupted the heptadmotif and stabilized the pre-fusion form. Furthermore, both HR1redesigns contained prolines, at positions 2 and 6 in the 8-residue loopand at position 8 in the 10-residue loop, which likely increased therigidity of the backbone. Of note, Asp 6 in HR1 redesign 9 is poised toform a salt bridge with Arg 579 of the neighboring HR1 helix,stabilizing the slightly turned loop. In conclusion, gp140 appears to behighly tolerant of the HR1 redesign, which greatly enhances proteinproduction efficiency without sacrificing overall structural integrity.

Example 4 Antigenic Profiling of HR1-Redesigend BG505 gp140 Trimers

The BG505 SOSIP.664 trimer represents a close mimic of the native spikein immune recognition by antibodies. Here we sought to investigatewhether HR1 redesign would affect Env trimer binding to bNAbs or affectbinding to non-NAbs using bio-layer interferometry (BLI) andimmunoglobulin G (IgG). Again, we studied trimers prepared using asimple GNL purification so we could more readily compare the basicproperties of different trimer constructs. BN-PAGE of SEC fractionsobtained from a Superdex 200 16/600 column following GNL purificationwas performed to facilitate selection of well-folded trimers forantigenic profiling. In this context, we also characterized the HR1redesign 1 by negative-stain EM. In the unliganded state, the 22 Åreconstruction displayed a morphology closely resembling that of theSOSIP trimer prepared using the same protocol. The agreement of crystaland EM structures further confirmed the integrity of HR1-redesignedtrimers prior to antigenic characterization.

First, we measured trimer binding to a panel of representative bNAbs. Weutilized V1V2 apex-directed, quaternary bNAbs PGDM1400, PGT145, and PG16to examine whether the trimeric structure with associated glycan shieldwas native-like. For PGDM1400, the HR1 redesigns 1 and 9 displayedfaster on- and off-rates than WT SOSIP, with a comparable KD (Koff/Kon)of 7 to 11 nM. A similar pattern was observed for PG16 and PGT145. ForVRC01, a representative of a class of CD4-binding site (CD4bs)-directedbNAbs, all three trimers showed nearly identical binding profiles,suggesting that the HR1 redesign had little effect on the presentationof this conserved site of vulnerability. A similar pattern was also seenfor NAb b12, which engages the CD4bs with a different angle of approachrelative to VRC01. For bNAbs targeting the V3 stem and surroundingglycans (PGT121, PGT128 and PGT135) and the high-mannose gp120 glycancluster (2G12), all three trimers showed identical binding profiles,indicating that these glycan epitopes remained intact upon HR1 redesign.Finally, we measured trimer binding to two bNAbs that recognizeconformational epitopes spanning regions in both gp120 and gp41. Allthree trimers bound strongly to PGT151 with a fast on-rate and a flatdissociation curve, with subtle differences observed in 35O22 bindingkinetics.

Next, we measured trimer binding to a panel of representative non-NAbs.All three tested trimers bound to CD4bs-specific MAbs, b6 and F105. TheHR1-redesigned trimers displayed weaker binding to F105 than did theSOSIP trimer, with a slightly faster off-rate detected for HR1redesign 1. However, no differences in kinetics were observed for b6.For two V3-specific MAbs, 19b and 447-52D, all three trimers showed fastassociation and slow dissociation, indicative of some V3 exposure thatwas confirmed by surface plasmon resonance (SPR) using the 2G12-purifiedSOSIP trimer. Previously, 19b was found to bind the SOSIP trimer byELISA, but only to a limited extent by EM. Nevertheless, the V3 exposuremay be minimized by conformational fixation as demonstrated recently forthe SOSIP trimer. We then tested two MAbs targeting the immunodominantepitopes in cluster I of gp41_(ECTO), F240 and 7B2. The SOSIP trimerappeared to bind both MAbs at a low level with a slight preference forF240. Interestingly, the two HR1 redesigns showed reduced binding toF240 and an almost negligible binding to 7B2, indicating a more closedor less flexible gp41_(ECTO). We also investigated the binding of twoCD4i MAbs, 17b and A32. All three trimers showed no binding to 17b inthe absence of sCD4, with the HR1 redesign 1 exhibiting only a minimallevel of A32 recognition although all trimers bound weakly to this MAb.

Overall, the two HR1 redesigns displayed broadly similar patterns intheir recognition by bNAbs with an exception of altered kinetics forapex-directed quaternary bNAbs. While all three trimers showed some V3exposure, the two HR1 redesigns appeared to shield non-neutralizinggp41_(ECTO) epitopes more effectively. The observed binding to non-NAbsmay be attributed to the use of IgG instead of Fab and a differentimmobilization strategy in the BLI experiment.

Example 5 Replacing the Furin Cleavage Site with Short Linkers

Sharma et al. recently reported a native-like, cleavage-independentgp140 trimer designated NFL (Cell Rep. 11, 539-550, 2015). In a separatestudy, Georgiev et al. replaced the cleavage site between gp120 and gp41with linkers of up to 20 residues designated sc-gp140 (J. Virol. 89,5318-5329, 2015). Although the presence of aberrant structures wasspeculated for sc-gp140 trimers with short linkers, the precise effectof cleavage site modification on gp140 folding and structure remainedunclear. Here, we addressed this critical issue in the context of theHR1 redesign 1 that had been validated both structurally andantigenically (FIGS. 2 and 3 ).

We first examined the outcome of replacing the cleavage site-containingregion (residues 500-519) with a redesigned connecting loop betweengp120 and gp41. The Cα distance between R500 and F519 is 16.8 Å,equivalent to a fully extended backbone of 4.4 residues. Ensemble-basedprotein design yielded a large pool of 8-residue loops connecting R500and F519 (FIG. 4 a ). Of note, this design strategy was ratheraggressive in that these loops may pack differently than the uncleavedWT sequence due to a 10-residue truncation in this region, and exclusionof the SOS mutation since A501 was now part of the region subjected toredesign (with the T605C mutation reversed). Similar to the HR1redesign, the 5 top-ranking designs (termed CST1-5, FIG. 4 b ) werecharacterized by SEC following transient expression in HEK293 F cellswithout furin followed by GNL purification. Overall, CST1-5 showedreduced trimer yield, as well as increased aggregates compared to theparent HR1 redesign, indicated by a higher shoulder left of the maintrimer peak in the SEC profiles. For all 5 CST redesigns. an extra bandwas observed in BN-PAGE analysis, suggesting the presence of anuncharacterized Env species in the produced proteins (FIG. 4 c ).

We next examined the effect of replacing the cleavage site (₅₀₈REKR₅₁₁)with a near full-length SGS linker (termed CSF). Interestingly, CSFdisplayed a notably reduced aggregate peak in the SEC profile comparedto CST1-5, which was further improved by adding back the SOS mutation(termed CSF-SOS). Similar to CST1-5, an extra band was observed for theCSF trimer in BN-PAGE analysis of trimer-containing fractions after SECon a Superdex 200 16/600 column, suggesting a common pattern associatedwith short cleavage site linkers. To identify this unknown Env species,we used negative-stain EM to obtain 3D reconstructions for the CSFtrimer. Remarkably, two distinct morphologies were observed for theunliganded trimer: one in the pre-fusion state (20 Å) similar to theSOSIP trimer and the other in a non-pre-fusion state (17 Å) that has notbeen previously reported. This non-pre-fusion trimer conformationcontains an extended gp41 (approximately 40-45 Å) and is termed “fusionintermediate” hereafter. The ˜20 Å EM reconstructions of PGV04-bound CSFtrimers in the two different states showed some unoccupied densitiesthat could not be interpreted at this resolution. By contrast, a singleconformation was observed for the EM reconstruction of the CSF-SOStrimer in both unliganded (21 Å) and PGV04-bound form (20 Å). Insummary, EM suggested that with short cleavage site linkers the CST andCSF trimers contain a fusion intermediate state that can be effectivelysuppressed by the SOS mutation.

We then tested the CSF and CSF-SOS trimer binding to a small panel ofbNAbs and non-NAbs. For bNAbs, we utilized PGDM1400, VRC01, and PGT151,which target the V1V2 apex, CD4bs, and gp120-gp41 interface,respectively. Both CSF and CSF-SOS trimers bound to PGDM1400 withsimilar kinetic profiles and affinities. However, due to the two mixedtrimer forms, CSF showed a reduced binding relative to CSF-SOS. CSF andCSF-SOS exhibited identical VRC01 binding profiles similar to that ofthe SOSIP trimer, suggesting that the CD4bs is equally accessible inthese trimers. For PGT151, CSF and CSF-SOS showed reduced binding with anotable off-rate, suggesting that the linker between gp120 and gp41 mayaffect PGT151 binding. Three non-neutralizing MAbs were also tested. CSFbound more strongly to CD4bs-directed F105 than CSF-SOS due to the mixedfusion intermediates. For the V3-directed 19b, both CSF trimersdisplayed similar binding profiles relative to the SOSP trimer and HR1redesign 1. By contrast, CSF showed enhanced binding to thegp41-directed F240 that was effectively reduced by the SOS mutation inCSF-SOS.

Example 6 Replacing the Furin Cleavage Site with Long Linkers

Based on our analysis thus far and the reports on NFL and sc-gp140trimers, we hypothesized that HR1 redesign combined with a long cleavagesite linker may overcome the tendency to form fusion intermediates andrender an uncleaved, pre-fusion optimized (UFO) trimer. To this end, wetested two trimers based on the HR1 redesign 1 and an NFL-like linker(2×G₄S). These two constructs, termed CSL and CSL-SOS, were transientlyexpressed in HEK293 F cells followed by GNL purification and SEC on aSuperdex 200 16/600 column. Both CSL trimers showed reasonable yieldswith similar SEC profiles to that of the CSF trimers. For CSL, althoughno extra bands were definitively identified on the BN gel, the trimerbands appeared to be more diffuse than those observed for CSL-SOS. Tofurther characterize their structures, we obtained EM reconstructionsfor the unliganded CSL and CSL-SOS trimers at 17 and 20 Å resolutions,respectively. The CSL trimer showed a somewhat different morphology thanof WT SOSIP, HR1 redesign 1, pre-fusion CSF and CSF-SOS trimers: thedensity of CSL trimer appeared to be narrower at the top of the trimerapex with additional densities pointing outwards and a wider bottomaround gp41. The overall shape of the CSL-SOS trimer was consistent withthat of the CSF-SOS trimer. The ˜20 Å reconstructions of PGV04-bound CSLand CSL-SOS trimers resembled that of the SOSIP trimer, indicative ofstabilization upon bNAb binding. Taken together, a long cleavage sitelinker can reduce the formation of fusion intermediates, likely at thecost of greater conformational variability, as suggested by EM.

We performed antigenic profiling for the CSL and CSL-SOS trimers usingthe same panel of bNAbs and non-NAbs as for the two HR1 redesigns. Forapex-directed bNAbs PGDM1400, PG16, and PGDM145, two CSL trimers showedsimilar binding kinetics and K_(D) values to those of HR1 redesign 1.For CD4bs-directed bNAb (VRC01), NAb b12, and glycan-reactive bNAbs(PGT121, PGT128, PGT135 and 2G12), the two CSL trimers showed nearlyidentical binding profiles to those of HR1 redesign 1 and the SOSIPtrimer. As expected, the most visible difference was found for bNAbsPGT151 and 35O22. For PGT151, which binds an epitope consisting of onegp120 and two adjacent gp41s in trimer, the cleaved HR1-redesignedtrimers and SOSIP trimer showed flat dissociation curves. However, theCSL trimers showed faster off-rates similar to those observed for theCSF trimers, indicating a consistent effect caused by cleavage sitelinkers. By contrast, for 35O22, which binds to gp120 and gp41 in asingle gp140 protomer, the off-rates appeared to becleavage-independent. MAbs F240 and 7B2 revealed less accessiblenon-neutralizing epitopes on gp41 for CSL-SOS but not for CSL,consistent with observations for the two CSF trimers.

In summary, an NFL-like long linker between gp120 and gp41 used incombination with an optimal HR1 redesign yielded an uncleaved gp140 thatretained the most desirable traits of a pre-fusion trimer. Our detailedanalysis of linker length also revealed complex consequences of cleavagesite modification. Thus, changing the linker length at the cleavage sitemust be carefully evaluated in each case in trimer immunogen design.

Example 7 A Generic HR1 Redesign to Stabilize Env Trimers of Diverse HIVStrains

Although cleavage site linkage might cause complications, the HR1redesign appeared to have an overall positive effect on trimer structureand antigenicity. In light of this finding, we revisited the HR1redesign strategy by examining the utility of a simple GS linker (FIG. 2a ). Such a “generic” HR1 linker (termed HR1-G), if proven successful,will not only confirm the role of this HR1 region in Env metastabilitybut also enable development of stable trimers for diverse HIV-1 strains.To this end, we tested the generic HR1 linker in the backgrounds ofclade-A BG505, clade-B JRFL, clade-C DU172.17, and a B′/C recombinantstrain CH115.12 (tier 3), with their SOSIP trimers included forcomparison. All trimer constructs were transiently expressed in HEK293 Fcells with furin, followed by GNL purification and SEC on a Superdex 20010/300 column. For the four strains studied, the generic HR1 linkershowed consistent improvement on trimer yield and purity (FIG. 2 b ).The most substantial improvement was observed for the clade-C strain: a46% increase of trimer peak relative to WT SOSIP with the aggregate anddimer/monomer peaks reduced by 34% and 37%, respectively. For thisclade-C strain, two top-ranking HR1 redesigns from ensemble-basedprotein design further increased the trimer peak by ˜50% with identicalSEC profiles to the generic HR1 redesign, HR1-G. The results thusindicate that the generic HR1 linker offers a general framework forstabilization of Env while further optimization of trimer properties canbe achieved by computational design in a strain-specific manner.

Example 8 Some Exemplified Methods for HR1 Redesigned HIV-1 Immunogen

Ensemble-based de novo protein design. We developed an ensemble-based denovo protein design method (FIG. 1 c ). Given the trimer structure (PDBID: 4TVP) and a specified loop length, a three-step design process wasundertaken: (1) an ensemble (1,000) of backbone conformations isgenerated to connect the two anchor residues using a torsion-space loopsampling algorithm⁴⁸; (2) for each backbone, a starting sequence isselected from a pool of 50 random sequences based on the RAPDF potentialand subjected to 500 steps of Monte Carlo simulated annealing (MCSA)with the temperature linearly decreasing from 300 to 10K; (3) thelowest-energy sequence for each backbone is recorded and allMCSA-derived designs are ranked based on energy at the completion of theprocess. The top 20 designs are manually inspected to facilitateselection of 5 candidates for experimental validation.

Antibodies for antigenic profiling. We utilized a panel of bNAbs andnon-NAbs to characterize the antigenicity of designed trimers. The bNAbs2G12 and b12 as well as MAbs F240, 7B2, 17b, and A32 were requested fromthe NIH AIDS Reagent Program.

Expression and purification of HIV-1 Env trimers. Env trimers weretransiently expressed in HEK293 F cells (Life Technologies, CA) exceptfor crystallographic analysis. Briefly, 293F cells were thawed andincubated with FreeStyle™ 293 Expression Medium (Life Technologies, CA)in the Shaker incubator at 37° C., with 120 rpm and 8% CO₂. When thecells reached a density of 2.0×10⁶/ml, expression medium was added toreduce cell density to 1.0×10⁶/ml for transfection withpolyethyleneimine (PEI) (Polysciences, Inc). For SOSIP andHR1-redesigned trimers, 800 μg of Env plasmid and 300 μg of furinplasmid in 25 ml of Opti-MEM transfection medium (Life Technologies, CA)was mixed with 5 ml of PEI-MAX (1.0 mg/ml) in 25 ml of Opti-MEM, whereasfor uncleaved trimers, 900 μg of Env plasmid was used without furin.After incubation for 30 min, the DNA-PEI-MAX complex was added to 1 L293F cells. Culture supernatants were harvested five days aftertransfection, clarified by centrifugation at 1800 rpm for 20 min, andfiltered using a 0.45 μm filters (Thermo Scientific). The Env proteinswere extracted from the supernatants using a Galanthus nivalis lectin(GNL) column (Vector Labs). The bound proteins were eluted with PBScontaining 500 mM NaCl and 1 M methyl-α-D-mannopyranoside and thenpurified by size exclusion chromatography (SEC) on a Superdex 200Increase 10/300 GL column for initial assessment and a HiLoad 16/600Superdex 200 PG column (GE Healthcare) for EM analysis and antigenicprofiling. Protein concentrations were determined using UV₂₈₀ absorbancewith theoretical extinction coefficients.

Blue Native (BN) PAGE. Env proteins were analyzed by blue nativepolyacrylamide gel electrophoresis (BN-PAGE) and stained with Coomassieblue. The protein samples were mixed with loading dye and loaded onto a4-12% Bis-Tris NuPAGE gel (Life Technologies). BN PAGE gels were run for2 hours at 150 V using NativePAGE™ running buffer (Life Technologies)according to the manufacturer's instructions.

Differential Scanning calorimetry (DSC). Thermal melting curves of SOSIPand HR1-redesigned trimers were obtained with a MicroCal VP-Capillarycalorimeter (Malvern). The SEC purified glycoproteins were bufferexchanged into 1×PBS and concentrated to 0.5-1 μM prior to analysis bythe instrument. Melting was probed at a scan rate of 90° C./hr. Dataprocessing including buffer correction, normalization, and baselinesubtraction were conducted using the standardized protocol from theOrigin 7.0 software.

Protein production and purification for crystallization. The twoHR1-redesigned trimers, as well as the 8ANC195 and PGT128 Fabs, wereproduced and purified as described in Kong et al., Acta Crystallogr.Sect. D-Biol. Crystallogr. 71, 2099-2108, 2015. Briefly, all constructswere cloned into the expression vector phCMV3. Fabs were transientlytransfected into mammalian Freestyle™ 293F cells, and trimers weretransiently transfected in in GnT1^(−/−) 293 S cells. After one week oftransfection, the supernatants of the antibody transfected cells wereharvested and purified using a Lambda or a Kappa Capture Select column(BAC BV) for PGT128 and 8ANC195 respectively before further purificationusing ion exchange chromatography and SEC. The supernatants of thetrimer transfected cells were purified using a 2G12 affinity columnfollowed by SEC. The trimer complexes used for crystallization trialswere prepared by mixing the trimer proteins with Fabs PGT128 and 8ANC195at a molar ratio of 1.0:1.2:1.2 at room temperature for 20 min. Thismixture was then deglycosylated using endoglycosidase H (EndoH) in 200mM NaCl, 50 mM sodium citrate, pH 5.5, for 37° C. for 1 hr following themanufacturer's protocol (New England Biolabs) before final purificationby SEC.

Protein crystallization and data collection. Two purified proteincomplexes containing Fabs PGT128 and 8ANC195 bound to HR1-redesignedtrimers were prepared for crystallization by buffer exchange into 50 mMNaCl, 20 mM Tris-HCl, pH 7.2. The complexes were then concentrated to 5mg/ml and passed through a 0.22 μm filter before crystal screening usingthe IAVI/JCSG/TSRI CrystalMation robot (Rigaku) at the JCSG. Similarlyto a previously described complex containing Fabs PGT128 and 8ANC195bound to SOSIP gp140 trimer, the HR-1 redesigned trimer/Fab complexescrystallized at 25° C. in 0.05 M lithium sulfate, 0.05 M sodium sulfate,20% (w/v) PEG 400 and 0.05 M Tris-HCl, pH 8.7 (JCSG Core Suitecondition: JCSGI A03, Qiagen). All crystals for X-ray data collectionwere cryoprotected by brief immersion in mother liquor supplemented with40% PEG 400 prior to flash-cooling in liquid nitrogen. For the HR1redesign 1 complex, diffraction data to 6.3 Å resolution were collectedat beamline 231D-B at the Advanced Photon Source, processed withHKL-2000, and indexed in space group I23 with 100% completeness and an<I>/<σ_(I)> of 2.3 in the highest resolution shell. For the HR1 redesign9 complex, diffraction data to 6.9 Å resolution were collected atbeamline 12-2 at the Stanford Synchrotron Radiation Lightsource,processed with HKL-2000, and indexed in space group I23 with 100%completeness and an <I>/<σ_(I)> of 1.3 in the highest resolution shell.Data collection and processing statistics are summarized in Table 1.

Structure determination and refinement. The structures of PGT128 and8ANC195 bound to the HR1-redesigned gp140 trimers were solved by themolecular replacement method using the Phaser software and a searchmodel consisting of a complex with SOSIP gp140 trimer bound to FabsPGT128 and 8ANC195 (PDBID: 5C7K). As described previously, refinementconsisted of alternating rounds of manual model building using Coot-0.7and automated refinement as implemented by the Phenix program. Given thelimited resolution of the datasets, grouped B-factor refinement for eachresidue was used. Furthermore, positional coordinate refinement wasenforced using a reference model set of restraints. The starting modelfor each automated refinement session in Phenix was defined as thereference model for that session. Finally, the model was minimallymodified except at the HR1 site of redesign. The final R_(cryst) andR_(free) values converged at 28.1% and 32.2%, and 28.4% and 32.2% forthe complex structures of HR1 redesigns 1 and 9, respectively. See Table1 for final refinement statistics. The Buried molecular surface areaswere analyzed with the Molecular Surface Package using a 1.7 Å proberadius and standard van der Waals radii. Fab residues were numberedaccording to Kabat nomenclature and gp140 was numbered using thestandard HXBc2 convention.

Electron microscopy sample preparation. The gp140 trimers alone, and incomplex with PGV04, were analyzed by negative stain EM. A 3 μL aliquotcontaining ˜0.02 mg/mL of the trimers was applied for 15 s onto acarbon-coated 400 Cu mesh grid that had been glow discharged at 20 mAfor 30 s, then negatively stained with 2% uranyl formate for 30 s. Datawere collected using a FEI Tecnai Spirit electron microscope operatingat 120 kV, with an electron dose of ˜30 e⁻/Å² and a magnification of52,000× that resulted in a pixel size of 2.05 Å at the specimen plane.Images were acquired with a Tietz 4 k×4 k TemCam-F416 CMOS camera usinga nominal defocus of 1000 nm and the Leginon package.

Electron microscopy data processing and image reconstruction. Particleswere picked automatically using DoG Picker and put into a particle stackusing the Appion software package. Initial, reference-free,two-dimensional (2D) class averages were calculated using particlesbinned by two via iterative multivariate statistical analysis(MSA)/multireference alignment (MRA) and sorted into classes. Particlescorresponding to trimers or to trimers bound to PGV04 were selected intoa substack and binned by two before another round of reference-freealignment was carried out using the iterative MSA/MRA and XmippClustering and 2D alignment programs. To analyze the quality of thetrimers (closed native-like, open native-like, and non-native), thereference free 2D class averages were examined by eye using the metricsdescribed in Pugach et al. J. Virol. 89, 3380-3395, 2015). An ab initiocommon lines model was calculated from reference-free 2D class averagesin EMAN2 imposing symmetry C3. This model was then refined against rawparticles for an additional 25 cycles using EMAN (Ludtke et al., J.Struct. Biol. 128, 82-97, 1999). The resolutions of the final modelswere determined using a Fourier Shell Correlation (FSC) cut-off of 0.5.

Binding Analysis by ELISA and Biolayer Light Interferometry (BLI). Thekinetics of trimer binding to bNAbs and non-NAbs was measured using anOctet Red96 instrument (fortéBio, Pall Life Sciences). All assays wereperformed with agitation set to 1000 rpm in fortéBIO 1× kinetic buffer.The final volume for all the solutions was 200 μl/well. Assays wereperformed at 30° C. in solid black 96-well plates (Geiger Bio-One). 5μg/ml of protein in 1× kinetic buffer was used to load an antibody onthe surface of anti-human Fc Capture Biosensors (AHC) for 300 s. A 60 sbiosensor baseline step was applied prior to the analysis of theassociation of the antibody on the biosensor to the Env trimer insolution for 200 s. A two-fold concentration gradient of trimer startingat 200 nM was used in a titration series of six. The dissociation of theinteraction was followed for 300 s. Correction of baseline drift wasperformed by subtracting the averaged shift recorded for a sensor loadedwith antibody but not incubated with trimer, or a sensor withoutantibody but incubated with trimer. Octet data were processed byfortéBio's data acquisition software v.8.1. Experimental data werefitted for V1V2 apex-directed bNAbs using a global fit 1:1 model todetermine the K_(D) values and other kinetic parameters.

Example 9 Ferritin Nanoparticles Presenting Trimeric V1V2 with aNative-Like Apex

The V1V2 region of gp120 ranges from 50 to 90 residues in length with 1in 10 residues N-glycosylated, forming a dense glycan shield on theHIV-1 Env. The V1V2-encoded glycan epitopes can be recognized by bNAbssuch as PG9/PG16, CH01-04, PGT141-145, and PGDM1400. Despite sequencevariation, a short segment centered at N160 defines the specificity formost known V1V2-specific bNAbs. The crystal structure of scaffolded V1V2in complex with PG9 has been determined for two clade C strains, CAP45and ZM109, revealing a Greek key motif with strands B and C harboringtwo critical glycans. The EM structures of PG9 and PGDM1400 in complexwith BG505 SOSIP.664 gp140 trimer indicated that these two bNAbs aredirected to the trimeric apex with different angles of approach asdescribed in, e.g., Julien et al., Proc. Natl. Acad. Sci. USA 110,4351-4356, 2013; and Sok et al., Proc. Natl. Acad. Sci. USA 111,17624-17629, 2014. In this study, we hypothesized that the threefoldaxes on ferritin nanoparticle can be utilized to present V1V2 in anative-like trimeric conformation found in the cryo-EM and crystalstructures of SOSIP trimer. To test this possibility, we designed twoconstructs based on the V1V2 of clade C ZM109: one containing all threedisulfide bonds (termed V1V2Ext) and a shortened version containing two(termed V1V2Sht), with both V1V2 sequences fused to the N-terminus (D5)of ferritin subunit (FR) (Table 2a) (See, e.g., Kanekiyo et al., Nature499, 102-106, 2013; and He et al., Sci. Rep. 5, 12501, 2015). Afterfitting the C-termini of trimeric V1V2 to the N-termini of ferritinsubunits around each threefold axis on the particle surface, structuralmodeling yielded Ca root-mean-square deviations (RMSDs) of 3.7 and 0.8 Åfor V1V2Ext-FR and V1V2Sht-FR, respectively. Further analysis revealed adense glycan surface for both nanoparticles with diameters of 16.6 and14.3 nm for V1V2Ext-FR and V1V2Sht-FR, respectively.

The two V1V2-ferritin constructs and the monomeric V1V2 were expressedtransiently in N-acetylglucosaminyltransferase I-negative (GnTI^(−/−))HEK293 S cells and purified using a Galanthus nivalis lectin (GNL)column followed by SEC on a Superdex 200 10/300 GL column. For bothV1V2-FR designs, the SEC profiles displayed a single peak indicative ofwell-formed nanoparticles, which were confirmed by blue nativepolyacrylamide gel electrophoresis (BN-PAGE). We then utilized negativestain EM to visualize the purified nanoparticles. Indeed, imaging by EMshowed homogeneous V1V2Ext-FR particles, which enabled the calculationof two-dimension (2D) class averages. Similar results were observed forV1V2Sht-FR particles, suggesting that homogeneity and purity areintrinsic to these nanoparticles despite their differences in the V1V2length and the number of disulfide bonds contained. However, thetrimeric V1V2 spikes appeared diffuse in the 2D class averages,indicative of some mobility. To probe the antigenicity of the V1V2 apex,we measured particle binding to PG9, which recognizes V1V2 in bothmonomeric and trimeric forms, and PGDM1400, which targets the apex ofthe SOSIP-like trimer conformation. Analysis of both nanoparticles byenzyme-linked immunosorbent assay (ELISA) showed enhanced bNAb bindingrelative to the monomeric V1V2, with PGDM1400 demonstrating preferentialbinding to V1V2Ext-FR. Using bio-layer interferometry (BLI) andimmunoglobulin G (IgG), we characterized the kinetics of V1V2 binding tobNAbs PG9 and PGDM1400 in monomeric and particulate forms. As expected,monomeric V1V2 bound to PG9 with low affinity and showed no binding toPGDM1400. By contrast, V1V2Ext-FR bound to both bNAbs with highaffinity, but different kinetics. For PGDM1400, V1V2Ext-FR showed anextremely fast on-rate, indicating a stable apex that can be readilyrecognized by this apex-directed bNAb. V1V2Sht-FR exhibited similarbinding kinetics with respect to V1V2Ext-FR, but with a reduced affinityfor PGDM1400 that suggests an adversary effect of the shortened V1V2 onthe apex structure and antigenicity consistent with ELISA.

Our results demonstrate that the V1V2 region of HIV-1 Env can bepresented in a native-like trimeric conformation on ferritinnanoparticles. Of note, this design strategy is likelystrain-independent, since nanoparticles were also observed forV1V2Ext-FR designed based on clade C CAP45. Overall, particulate displayof trimeric V1V2 substantially improved its recognition by apex-directedbNAbs, suggesting that V1V2 nanoparticles may provide promisingalternatives to gp140 trimers and focus B cell responses to thisquaternary epitope.

Example 10 Ferritin Nanoparticles Presenting Trimeric V1V2 with aNative-Like Apex

A 60-meric LS nanoparticle presenting an engineered gp120 core lackingvariable loops (V1V2 and V3) and inner domain has been used to targetgermline precursors of VRC01, a CD4-binding site (CD4bs)-directed bNAb.However, structures of BG505 SOSIP trimer in complex with theVRC01-class bNAb PGV04 revealed that glycans on the neighboring gp140protomer are also involved in CD4bs recognition, suggesting an angle ofapproach constrained by the trimeric context. The importance of trimerconstraints for HIV-1 neutralization was further demonstrated in humanIg knock-in mice, in which only BG505 SOSIP trimer, but not eOD-LSnanoparticle, elicited NAb responses. Based on these previous studiesand the results of V1V2 nanoparticles, we hypothesized that ferritinnanoparticle can be used to present full-length gp120 and expose all itsencoded bNAb epitopes in their native-like conformations as does theSOSIP gp140 trimer. This design strategy may generate alternativeimmunogens lacking the complications intrinsic to the gp140 trimerscontaining metastable gp41. To test this possibility, we designed threeferritin fusion constructs based on clade A BG505 gp120: gp120Ext-FR andgp120Sht-FR contained different lengths of gp120, while gp120SS-FRincorporated an additional disulfide bond aimed to stabilize the gp120termini (Table 2b). In the case of gp120Ext-FR, structural modelingrevealed a nearly perfect superposition of gp120 C-terminus (G495) andferritin N-terminus (D5) around each threefold axis on the particlesurface, with a Ca RMSD of 1.9 Å and a diameter of 26.2 nm for theresulting nanoparticle.

All three gp120-ferritin constructs and the monomeric gp120 wereexpressed transiently in HEK293 F cells. The secreted proteins werepurified using a GNL column followed by SEC on a Superose 6 10/300 GLcolumn. Among the three chimeric constructs, gp120Sht-FR showed the mostpronounced particle peak in the SEC profile. Of note, no particle peakwas observed for gp120SS-FR, suggesting misfolding of the mutant gp120.BN-PAGE revealed high molecular weight (m.w.) bands for both gp120Ext-FRand gp120Sht-FR, corresponding to fully assembled nanoparticles. We thenemployed negative stain EM to visualize the purified gp120-FRnanoparticles. Homogeneous particles were observed for both gp120Ext-FRand gp120Sht-FR, with 2D class averages calculated for the latter. Sincegp120Sht-FR displayed more efficient particle assembly, cryo-EM wasutilized to further characterize the nanoparticles, showing a particlesurface decorated with gp120 spikes.

To assess the antigenicity of gp120 nanoparticles, we measured thekinetics of particle binding to a panel of representative bNAbs andnon-NAbs. We first tested apex-directed bNAbs PG16 and PGDM1400. Asexpected, gp120 monomer exhibited only minimal binding to PG16 andalmost undetectable binding to PGDM1400. By contrast, gp120nanoparticles showed substantially enhanced binding to both bNAbs withsub-nanomolar affinities, with gp120Sht-FR slightly outperforminggp120Ext-FR. This confirmed that three gp120s around each threefold axison a ferritin nanoparticle can indeed form SOSIP-like trimerconformations. For CD4bs-directed VRC01, both nanoparticles displayed anincreased on-rate with flat dissociation curves. A similar trend wasobserved for NAb b12, which targets the same site. The avidity effectresulting from multivalent display was most pronounced for bNAb PGT121,which targets the V3 base and surrounding glycans: while monomeric gp120bound to PGT121 at a lower level with a fast off-rate, both gp120nanoparticles showed enhanced binding with faster on-rates and flatdissociation curves. We then measured particle binding tonon-neutralizing MAbs. For F105, which prefers an open gp120conformation, monomeric gp120 displayed rapid on- and off-rates, whereasnanoparticles showed slower on- and off-rates that may result from thesteric hindrance caused by the dense display of gp120 trimers. However,gp120 nanoparticles did show enhanced recognition by the V3-specific 19bin comparison to monomeric gp120, which may be minimized byconformational fixation as recently demonstrated for the SOSIP trimer.Lastly, gp120-FR nanoparticles showed almost negligible binding to theCD4i MAb 17b in contrast to a notable recognition of monomeric gp120 bythis MAb.

Example 11 60-Meric Nanoparticles Presenting Trimeric gp120

We also investigated whether 60-meric nanoparticles could be utilized topresent trimeric gp120. We selected two thermostable 60-mers withdistinct structural features—LS (Zhang et al., J. Mol. Biol. 306,1099-1114, 2001) and E2p (Izard et al., Proc. Natl. Acad. Sci. USA 96,1240-1245, 1999)—to examine this possibility. Compared to the 12.2-nmdiameter of ferritin, LS is only slightly larger in size, with adiameter of 14.8 nm. Structural modeling of the gp120Sht-LS nanoparticleindicated that the LS surface would be covered entirely by 20 trimericgp120 spikes with an estimated diameter of 28.7 nm. Following transientexpression in HEK293 F cells and GNL purification, the secreted proteinwas analyzed by SEC. However, no particle peak was observed in the SECprofile of this LS construct. Consistently, BN-PAGE showed a predominantpentamer band, which was confirmed by the negative stain EM analysis. Inbrief, our results indicate that the relatively small LS nanoparticlemay not be optimal for displaying full-length gp120 trimeric spikes.

We next examined E2p, which is a hollow dodecahedron with a diameter of23.2 nm and 12 large openings separating the threefold vertices on theparticle surface. Structural modeling yielded a gp120Sht-E2pnanoparticle with a diameter of 37.6 nm (Table 2c), which is close tothe optimal size for direct uptake by DCs. The HEK293 F-expressed,GNL-purified gp120Sht-E2p protein was analyzed by SEC on a Superose 610/300 GL column, which showed a distinctive high m.w. peakcorresponding to the chimeric E2p particles. Concordantly, BN-PAGEshowed a concentrated nanoparticle band on the gel, with a lighter bandof low m.w. suggestive of dissociated gp120-E2p trimer. We then utilizednegative stain EM to characterize the assembly of this 60-mer andobserved large, homogeneous nanoparticles. The 2D class averagesrevealed hollow protein cages resembling the crystal structure. However,while the EM analysis validated the robustness of E2p as a nanoparticleplatform for large and complex antigens such as trimeric gp120, it alsoshowed unexpected 2D class averages lacking the gp120 spikes. It wasunclear from the EM analysis alone whether the trimeric gp120 spikesformed but remained mobile due to the large spacing, or if the threegp120s around each threefold axis failed to assemble into a stabletrimer. To address this issue, we measured E2p particle binding to asmall panel of bNAbs and non-NAbs by BLI. Remarkably, gp120Sht-E2p boundto the apex-directed bNAbs PG16 and PGDM1400 with sub-picomolaraffinities. The fast on-rate and flat dissociation curves indicatednative-like apexes resembling that of the SOSIP trimer but withadditional advantage of avidity. Similar to the case of gp120-ferritinnanoparticles, we observed increased recognition of the CD4bs and V3base by bNAbs VRC01 and PGT121, as well as NAb b12. For non-neutralizingMAbs, gp120Sht-E2p bound to the CD4bs-specific Mab F105 and V3-specific19b at a level similar to gp120Sht-FR, suggesting a common featureshared by gp120 nanoparticles irrespective of the size. Lastly, onlyminimal binding was observed for the CD4i MAb, 17b.

As the receptor-binding protein of HIV-1 Env, gp120 has been extensivelystudied as a vaccine immunogen, but is now considered suboptimal due tothe exposure of a non-neutralizing face that is buried within the nativespike. Our results indicate that display of full-length gp120 withferritin and E2p nanoparticles can restore the native-like trimerconformation in the absence of gp41. With SOSIP-like antigenicity andvariations in particle size and surface spacing, these nanoparticlesprovide versatile platforms to investigate gp120-based HIV-1 vaccines.

Example 12 Ferritin Nanoparticles Presenting Stabilized Gp140 Trimers

The design and immunogenicity of a ferritin nanoparticle presenting theBG505 SOSIP gp140 trimer were recently reported (Sliepen et al.,Retrovirol. 12:82, 2015). The analysis of this gp140 nanoparticle byELISA showed notably reduced binding to the apex-directed bNAbs PG9 andPGT145, and to a bNAb directed to the gp120-gp41 interface, PGT151. Thisis somewhat surprising given the antigenic profiles we observed forgp120 nanoparticles using BLI. In this study, we sought to approach theferritin display of gp140 trimer with a new stabilization designcontaining a modified HR1 bend (residues 548-568, termed HR1 redesign1), and a detailed analysis of linker length and gp41 truncation. Thegp140 constructs tested here included: a gp140 truncated at position 664(gp140.664), gp140.664 with a 10-residue linker (gp140.664-10aa), and agp140 truncated at position 681 to include MPER with the same linker(gp140.681-10aa) (Table 2d). Structural modeling of the HR1-redesignedgp140-ferritin particles indicated well-separated trimer spikes withdiameters of 30.1, 35.7, and 40.1 nm for gp140.664-FR,gp140.664-10aa-FR, and gp140.681-10aa-FR, respectively.

Since contaminant Env species cannot be eliminated during particleassembly, the purity of gp140 trimer will have a significant impact onthe quality of gp140 nanoparticles. To illustrate this problem, wecompared the SEC profiles of the BG505 SOSIP trimer and anHR1-redesigned gp140 trimer, which showed substantial differences inaggregate and dimer/monomer peaks, as indicated by the UV absorbance at280 nm. All gp140-ferritin nanoparticles, including a SOSIP-baseddesign, were transiently co-expressed with furin in HEK293 F cells andpurified using GNL followed by SEC on a Superose 6 10/300 GL column.Using negative stain EM, we first confirmed the assembly ofSOSIP-ferritin nanoparticles. The GNL-purified gp140-ferritin proteinscontaining the HR1 redesign 1 exhibited similar SEC profiles with hightrimer and dimer/monomer peaks relative to the particle-containing peakat 8.5-10.5 mL. For gp140.664-10aa-FR, which contained morenanoparticles, EM revealed an unknown protein species with a hexagonalstructure mixed with aggregates and well-assembled particles. To improvethe particle purity, we investigated the utility of Capto Core 700column, which has been used for VLP purification. After purificationusing Capto Core 700 and GNL columns, gp140.664-10aa-FR showed reducednon-particle peaks in the SEC profile. Consistently, higher-quality EMimages were obtained with the hexagonal structures still present,indicating that an HIV-1 Env-specific purification method is required.To this end, we examined the combined use of Capto Core 700 and 2G12affinity columns, the latter of which has been widely used to purifySOSIP trimers. Overall, gp140.664-10aa-FR remained the best performer,showing a more visible particle peak in the SEC profile with a reducedtrimer peak and no dimer/monomer peak. For all three constructs, theparticle-containing fractions were analyzed by BN-PAGE, which showedhigh m.w. bands corresponding to fully assembled gp140 nanoparticlesrelative to the individual trimer. Of note, these bands are consistentwith the SEC profile and the estimated shift from the gp120-ferritinparticle bands, in contrast to the Sliepen et al. report. Forgp140.664-10aa-FR, homogeneous nanoparticles with visible spikes on thesurface were observed in negative stain EM. The stability ofgp140.664-10aa-FR nanoparticles was confirmed by EM analysis of a samplethat had been frozen and thawed. We also observed well-formednanoparticles for gp140.664-FR and gp140.681-10aa-FR in negative stainEM. Taken together, our results validated the assembly of gp140-ferritinnanoparticles, and highlighted the importance of proper purification andcharacterization in the development of gp140-based nanoparticles.

We characterized the antigenic profiles of gp140 nanoparticles using BLIand a panel of representative bNAbs with the HR1-redesigned trimerincluded as a control. We first utilized V1V2 apex-directed bNAbsPGDM1400, PGT145, and PG16 to probe the apex of gp140 trimers displayedon the particle surface. Remarkably, all gp140 nanoparticles showedsub-picomolar affinities compared to the individual trimer, with flatdissociation curves due to avidity. For bNAb PGT121, which targets theV3 epitope centered at N332, gp140 nanoparticles showed similar bindingprofiles to that of the HR1-redesigned trimer with an increased on-rate.For CD4bs-directed bNAb VRC01, gp140-ferritin particles displayed slowassociation similar to gp120-ferritin particles. For bNAbs PGT151 and35O22 that target the gp120-gp41 interface, we found enhancedrecognition, but with different kinetics. For PGT151, a faster on-ratewas observed with an unchanged dissociation pattern. By contrast, anincreased on-rate was observed for 35O22 that was accompanied by a rapiddissociation. In brief, all three gp140 nanoparticles showed improvedrecognition by bNAbs except for VRC01, suggesting that the crowdedsurface display of gp140 trimers may have the most significant impact onthe VRC01-class bNAbs that bind the CD4bs with a restricted angle ofapproach.

We next measured the binding kinetics of gp140 nanoparticles tonon-NAbs. Notably, reduced binding was observed for the CD4bs-specificMAbs F105 and b6, with a more significant reduction seen for b6. In thecase of V3-specific MAb 19b, a slower association was observed for gp140nanoparticles compared to trimer and gp120 nanoparticles, suggesting aminimized V3 exposure due to the dense display of gp140 trimers. For MAbF240, which targets the immunodominant epitopes in cluster I of gp41,gp140 nanoparticles exhibited undetectable binding compared to theresidual binding observed for the HR1-redesgined trimer. For both gp140trimer and gp140 nanoparticles, binding to CD4i MAbs 17b and A332 wasnot detected. Lastly, we utilized MPER-directed bNAbs 4E10 and 10E8 toprobe this gp41 epitope in the context of gp140.681-10aa-FR.Surprisingly, although this nanoparticle bound to 4E10 with a rapidon-rate and flat dissociation curve, it showed only minimal binding to10E8, which recognizes a conformational epitope spanning beyond the 4E10binding site. As revealed by structural modeling, since MPER is proximalto the ferritin surface with a distance of ≥10 nm from the outersurface, steric hindrance may have a more significant impact on bNAbssuch as 10E8 that select for certain epitope conformations.

Our analyses revealed critical characteristics of the designedgp140-ferritin nanoparticles. In contrast to the Sliepen et al. report,the antigenic profiling by BLI clearly demonstrated enhanced recognitionby bNAbs and reduced binding to non-NAbs. The results suggest that thesegp140 nanoparticles may be superior immunogens to individual gp140trimers in eliciting robust B cell responses towards the bNAb epitopes.Of note, the intrinsic purity of HR1-redesigned trimer has played anindispensable role in the production of nanoparticles with native-likegp140 trimers that demonstrated SOSIP-like antigenic profiles withnotably reduced binding to non-NAbs.

Example 13 A 60-Meric E2p Nanoparticle Presenting Stabilized Gp140Trimer

Based on the promising results of gp140-ferritin nanoparticles, weinvestigated the particulate display of gp140 trimer on two 60-mers, LSand E2p. Given the small size of LS, we first designed a constructcontaining a 10-residue linker between the C-terminus of LS subunit andN-terminus of gp140 (Table 2e). Structural modeling of LS-10aa-gp140.664yielded an estimated diameter of 39.2 nm. Following furin co-expressionin HEK293 F cells and GNL purification, the chimeric protein wasanalyzed by SEC and negative stain EM, in which well-formednanoparticles were not identified. We then examined the utility of60-meric E2p nanoparticle by fusing the BG505 gp140.664 containing aredesigned HR1 bend to the N-terminus of E2p core subunit (Table 2e).Structural modeling indicated that the gp140.664-E2p nanoparticle, 41.5nm in diameter, can expose all bNAb epitopes except for MPER. Thegp140.664-E2p construct was co-transfected with furin in HEK293 F cellsfollowed by the GNL purification and SEC on a Superose 6 10/300 GLcolumn. Although the overall expression was low, the highest peak in theSEC profile corresponded to the large E2p nanoparticles, which wasfurther confirmed by the SEC analysis of 2G12-purified sample. Apossible explanation for the improved efficiency in particle assembly isthat the association of three gp41 subunits can facilitate gp140trimerization and E2p assembly simultaneously. BN-PAGE showed a band onthe top of the gel characteristic of high m.w. nanoparticles.Homogeneous gp140.664-E2p nanoparticles with a dense layer of trimerspikes were observed from the EM analysis. Our results thus indicatethat gp140.664-E2p can form homogenous, VLP-size nanoparticles withdesired structural regularity of gp140 trimers poised for immunerecognition.

We then characterized the antigenicity of gp140.664-E2p nanoparticleusing a panel of bNAbs. For apex-directed PG16 and PGDM1400,gp140.664-E2p showed a slow on-rate with flat dissociation curves andless than picomolar affinities. For CD4bs-directed VRC01, gp140.664-E2pshowed notably reduced binding and flat dissociation curves reminiscentof the gp140-ferritin nanoparticles, but weaker. For bNAb PGT121, whichbinds to a glycan epitope at the V3 stem, we observed trimer-likekinetics with slightly reduced on-rate. For PGT151 and 35O22,gp140.664-E2p exhibited binding profiles similar to those of thegp140-ferritin nanoparticles. Overall, gp140.664-E2p showed reducedrecognition by the bNAbs tested in this analysis, with the most visiblechange observed for VRC01 and the least for PGT151. This result raisedthe possibility that non-native gp140 trimer conformations weredisplayed on the E2p nanoparticle. To address this critical issue, wetested particle binding to a panel of non-NAbs. For CD4bs-specific MAbF105, we observed weakened binding with a more rapid dissociationrelative to the individual trimer. Furthermore, to our surprise, 19bbinding revealed a notably reduced V3 exposure, as opposed to theenhancement observed for all other gp120 and gp140 nanoparticles. Thegp140.664-E2p nanoparticle bound to the CD4i MAb 17b at a minimal level,consistent with the previous observation for gp140-ferritinnanoparticles. For gp41-specific MAb F240, however, gp140.664-E2p showedslightly increased binding relative to the trimer and gp140-ferritinnanoparticles. Further analysis revealed an approximately 9 Å edge thatmade up the threefold vertices for E2p compared to a 20 Å edge forferritin, suggesting that a short linker might have caused some strainin the gp41-E2p connecting region, and consequently a less favorablegp41 conformation.

The gp140.664-E2p nanoparticle, with an optimal size (40-50 nm) for DCupdate, may be more advantageous than gp140-ferritin nanoparticles ineliciting strong and sustained B cell responses. Given the efficientassembly of gp140.664-E2p nanoparticles, it is possible that theparticulate display of gp140.681 can be achieved using E2p, and warrantsfurther investigation. In summary, our characterization of gp140.664-E2pnanoparticle has provided an important step towards the development ofhigh-valency, VLP-like HIV-1 vaccines.

TABLE 1 X-ray crystallographic data collection and refinementstatistics. HR1 redesign 1 + HR1 redesign 9 + Fabs 8ANC195 and Fabs8ANC195 and Data collection PGT128 PGT128 X-ray Source APS 23ID-D SSRL12-2 Wavelength (Å) 1.033 0.980 Space group I23 I23 Unit cell parametersa = b = c = 266.3 Å a = b = c = 262.0 Å α = β = γ = 90.0° α = β = γ =90.0° Resolution (Å) 50.0-6.30 (6.52-6.30) ^(a) 50.0-6.90 (7.15-6.90)^(a) Observations 104,666 96,139 Unique reflections 6,914 (685) ^(a)  5,022 (496) ^(a)   Redundancy 15.1 (15.7) ^(a) 19.1 (20.3) ^(a)Completeness (%) 100.0 (100.0) ^(a) 100.0 (100.0) ^(a) <I/σ_(I)>^(b)17.9 (2.3) ^(a)  15.5 (1.3) ^(a)  R_(sym) ^(c) 0.10 (2.09) ^(a) 0.16(4.21) ^(a) R_(pim) ^(c) 0.04 (0.54) ^(a) 0.05 (0.81) ^(a) CC_(1/2) 0.51^(a) 0.33 ^(a) Refinement statistics Resolution (Å) 40.14-6.31(6.79-6.31) ^(a) 47.83-6.92 (7.61-6.92) ^(a) Reflections (work) 6,208(1,236) ^(a) 4,519 (1,135) ^(a) Reflections (test) 684 (139) ^(a) 492(109) ^(a) R_(cryst) (%) ^(c) 28.1 28.4 R_(free) (%) ^(d) 32.2 32.2Average B-value (Å²) 350 292 Wilson B-value (Å²) 356 407 RMSD from idealgeometry Bond length (Å) 0.004 0.004 Bond angles (°) 0.841 0.882Ramachandran statistics (%) ^(f) Favored 95.1 95.2 Outliers 0.2 0.1 PDBID ^(a) Numbers in parentheses refer to the highest resolution shell.^(b)Calculated as average(I)/average(σI) ^(c) R_(sym) = Σ_(hkl)Σ_(i) |I_(hkl, i) − <I_(hkl)> |/Σ_(hkl)Σ_(i)I_(hkl, I), where I_(hkl, i), isthe scaled intensity of the i^(th) measurement of reflection h, k, l,<I_(hkl)> is the average intensity for that reflection, and n is theredundancy. R_(pim), is a redundancy-independent measure of the qualityof intensity measurements. R_(pim) = Σ_(hkl) (1/(n − 1))^(1/2) Σ_(i) |I_(hkl, i) − <I_(hkl)> |/Σ_(hkl) Σ_(i) I_(hkhl, I), where I_(hkl, i) isthe scaled intensity of the i^(th) measurement of reflection h, k, l, <I_(hkl) > is the average intensity for that reflection, and n is theredundancy. ^(d) R_(cryst) = Σ_(hkl) | F_(o) − F_(c) |/Σ_(hkl) | F_(o) |× 100 ^(e) R_(free) was calculated as for R_(cryst), but on a test setcomprising 10% of the data excluded from refinement. ^(f) These valueswere calculated using MolProbity.

TABLE 2Amino acid sequences of HIV-1 trimer-presenting nanoparticles.^(a)Construct name Amino acid sequencea. V1V2 trimer-presenting ferritin nanoparticles ZM109 V1V2Ext-FR[PCVKLTPLCVTLNCTSPAAHNESETRVKHCSFNITTDVKDRK (SEQ ID NO: 29)QKVNATFYDLDIVPLSSSDNSSNSSLYRLISCNTSTITQACP]ASGDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS ZM109 V1V2Sht-FR[ACVTLNCTSPAAHNESETRVKHCSFNITTDVKDRKQKVNATF (SEQ ID NO: 30)YDLDIVPLSSSDNSSNSSLYRLISCA]ASGDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNEN HGLYLADQYVKGIAKSRKSCAP45 V1V2Ext-FR [PCVKLTPLCVTLRCTNATINGSLTEEVKNCSFNITTELRDKKQ(SEQ ID NO: 31) KAYALFYRPDVVPLNKNSPSGNSSEYILINCNTSTITQACP]ASGDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKSb. gp120 trimer-presenting ferritin nanoparticles BG505 gp120Ext-FR[GVPVWKDAETTLFCASDAKAYDTEKHNVWATHACVPTDPN (SEQ ID NO: 32)PQEIHLENVTEEFNMWKNNMVEQMHTDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNVVRSELYKYKVVKIEPLG]ASGDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNE NHGLYLADQYVKGIAKSRKSBG505 gp120Sht-FR [GVWKDAETTLFCASDAKAYDTEKHNVWATHACVPTDPNPQ(SEQ ID NO: 33) EIHLENVTEEFNMWKNNMVEQMHTDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEG]ASGDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHG LYLADQYVKGIAKSRKSBG505 gp120SS-FR [GWCDAETTLFCASDAKAYDTEKHNVWATHACVPTDPNPQEI(SEQ ID NO: 34) HLENVTEEFNMWKNNMVEQMHTDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVCIG]ASGDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS (SEQ ID NO: 34)c. gp120 trimer-presenting LS and E2p nanoparticles BG505 gp120Sht-LS[GVWKDAETTLFCASDAKAYDTEKHNVWATHACVPTDPNPQ (SEQ ID NO: 35)EIHLENVTEEFNMWKNNMVEQMHTDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEG]ASGMQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDCIVRHGGREEDITLVRVPGSWEIPVAAGELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLANL ALELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEM ANLFKSLR BG505 gp120Sht-E2p[GVWKDAETTLFCASDAKAYDTEKHNVWATHACVPTDPNPQ (SEQ ID NO: 36)EIHLENVTEEFNMWKNNMVEQMHTDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEG]ASGAAAKPATTEGEFPETREKMSGIRRAIAKAMVHSKHTAPHVTLMDEADVTKLVAHRKKFKAIAAEKGIKLTFLPYVVKALVSALREYPVLNT

IDDETEEIIQ KHYYNIGIAADTDRGLLVPVIKHADRKPIFALAQEINELAEKARDGKLTPGEMKGASCTITNIGSAGGQWFTPVINHPEVAILGIGRIAEKPIVRDGEIVAAPMLALSLSFDHRMIDGATAQKALNHIK RLLSDPELLLMd. gp140 trimer-presenting ferritin nanoparticles^(b) BG505 gp140.664-FR[AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVW (SEQ ID NO: 37)ATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTV QARNLLSG

VWGIKQLQARVLAVERYLRDQQL LGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD]ASGDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELI GNENHGLYLADQYVKGIAKSRKSBG505 gp140.664-10aa- [AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWFR (SEQ ID NO: 38) ATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTV QARNLLSG

VWGIKQLQARVLAVERYLRDQQL LGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD] GSGSGSGSGS ASGDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS BG505 gp140.681-10aa-[AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVW FR (SEQ ID NO: 39)ATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTV QARNLLSG

VWGIKQLQARVLAVERYLRDQQL LGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALDKWASLWNWFDI TNWLWYIRA] GSGSGSGSGSASGDIIKLLNEQVNKEMQSSNLY MSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYL ADQYVKGIAKSRKSe. gp140 trimer-presenting LS and E2p nanoparticles BG505 LS-10aa-MQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDCIVRHGG gp140.664 (SEQ IDREEDITLVRVPGSWEIPVAAGELARKEDIDAVIAIGVLIRGATP NO: 40) HFDYIASEVSKGLANL

LELRKPITFGVITADTLEQAIERAGTK HGNKGWEAALSAIEMANLFKSLR GSGSGSGSGS ASG[AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNL LSG

VWGIKQLQARVLAVERYLRDQQLLGIWGC SGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD] BG505 gp140.664-E2p[AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVW (SEQ ID NO: 41)ATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTV QARNLLSG

VWGIKQLQARVLAVERYLRDQQL LGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD]ASGAAAKPATTEGEFPETREKMSGIRRAIAKAMVHSKHTAPHVTLMDEADVTKLVAHRKKFKAIAAEKGIKLTFLPYVVKALVSALREYPVLNT

ID DETEEIIQKHYYNIGIAADTDRGLLVPVIKHADRKPIFALAQEINELAEKARDGKLTPGEMKGASCTITNIGSAGGQWFTPVINHPEVAILGIGRIAEKPIVRDGEIVAAPMLALSLSFDHRMIDGATAQ KALNHIKRLLSDPELLLM ^(a)Foreach construct, the HIV-1 antigen is shown in brackets with themutations shown in underlined font. Mutations in the nanoparticlesequence aimed to remove N-linked glycosylation sites are shown inbold/underlined font. The enzymatic site (ASG) between HIV-1 antigen andparticle subunit is shown in italic font. ^(b)The gp140 sequencescontain a redesigned heptad repeat 1 (HR1) region that has been found tosignificantly improve trimer yield and purity while retaining theSOSIP-like structure and antigenicity. The modified HR1 region is shownin italic/bold font, and the 10 residue GS linker (SEQ ID NO: 42) isshown in italic/underlined font. A leader sequence“MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGAR” (SEQ ID NO: 43) is used for allgpl40 nanoparticle constructs.

Example 14 Some Exemplified Methods for HIV-1 Trimer PresentingNanoparticles

Nanoparticle design and modeling. A Perl script was developed to (1)search for three-fold vertices on the surface of a given nanoparticle orVLP, (2) superpose the C-termini of trimeric HIV-1 antigen ontoN-termini of three particle subunits around each three-fold axis on theparticle surface, and (3) generate XYZ coordinates of thetrimer-presenting particle with the diameter and other structuralparameters calculated at the completion of the process. The particlemodel obtained was visualized using UCSF Chimera for manual inspectionand selection of proper linkers.

Antibodies for antigenic profiling. We utilized a panel of bNAbs andnon-NAbs to characterize the antigenicity of designed trimers. The bNAbs2G12 and b12 as well as MAbs F240, 7B2, 17b, and A32 were requested fromthe NIH AIDS Reagent Program. Other bNAbs and non-NAbs were obtainedelsewhere.

Expression and purification of HIV-1 Env antigens and nanoparticles.Trimers and trimer-presenting nanoparticles were transiently expressedin HEK293 F cells (Life Technologies, CA), with monomeric V1V2 and V1V2nanoparticles transiently expressed in HEK293 S cells. Briefly, HEK293F/S cells were thawed and incubated with FreeStyle™ 293 ExpressionMedium (Life Technologies, CA) in a Shaker incubator at 37° C., with 120rpm and 8% CO₂. When the cells reached a density of 2.0×10⁶/ml,expression medium was added to reduce cell density to 1.0×10⁶/ml fortransfection with polyethyleneimine (PEI) (Polysciences, Inc). For gp140nanoparticles, 800 μg of fusion protein plasmid and 300 μg of furinplasmid in 25 ml of Opti-MEM transfection medium (Life Technologies, CA)was mixed with 5 ml of PEI-MAX (1.0 mg/ml) in 25 ml of Opti-MEM; whereasfor V1V2 and gp120 nanoparticles, 900 μg of chimeric Env plasmid wasused without furin. After incubation for 30 min, the DNA-PEI-MAX complexwas added to 1 L 293F/S cells. Culture supernatants were harvested fivedays after transfection, clarified by centrifugation at 1800 rpm for 22min, and filtered using 0.45 μm filters (Thermo Scientific). Theproteins were extracted from the supernatant using a Galanthus nivalislectin (GNL) column (Vector Labs). The bound proteins were eluted withPBS containing 500 mM NaCl and 1 M methyl-α-D-mannopyranoside andpurified by size exclusion chromatography (SEC) on a Superdex 200Increase 10/300 GL column or a Superose 6 10/300 GL column (GEHealthcare). Protein concentrations were determined using UV₂₈₀absorbance with theoretical extinction coefficients. For gp140-ferritinnanoparticles, Capto 700 Core column (GE Healthcare) and 2G12 affinitycolumn⁶ were used to improve particle purity.

Blue Native (BN) PAGE. HIV-1 trimer-presenting nanoparticles wereanalyzed by blue native polyacrylamide gel electrophoresis (BN-PAGE) andstained using Coomassie blue. The protein samples were mixed withloading dye and loaded onto a 4-12% Bis-Tris NuPAGE gel (LifeTechnologies). BN-PAGE gels were run for 2 hours at 150 V usingNativePAGE™ running buffer (Life Technologies) according to themanufacturer's instructions.

Electron microscopy sample preparation and data processing. The purifiednanoparticles were analyzed by negative stain EM. A 3 μL aliquotcontaining ˜0.01 mg/mL of the sample was applied for 15 s onto acarbon-coated 400 Cu mesh grid that had been glow discharged at 20 mAfor 30 s, then negatively stained with 2% uranyl formate for 45 s. Datawere collected using a FEI Tecnai Spirit electron microscope operatingat 120 kV, with an electron dose of ˜30 e⁻/Å² and a magnification of52,000× that resulted in a pixel size of 2.05 Å at the specimen plane.Images were acquired with a Tietz 4 k×4 k TemCam-F416 CMOS camera usinga nominal defocus of 1000 nm and the Leginon package. The nanoparticleswere picked automatically using DoG Picker and put into a particle stackusing the Appion software package. Reference-free, two-dimensional (2D)class averages were calculated using particles binned via the iterativemsa/mra Clustering 2D Alignment and IMAGIC software systems and sortedinto classes.

Binding Analysis by Enzyme-Linked Immunosorbend Assay (ELISA). Costar™96-well assay plates (Corning) were coated with V1V2 antigens overnightat 4° C. The wells were washed once with PBS+0.05 Tween 20, and thenincubated with 150 μl of blocking buffer (PBS with 5% w/v dry milk) perwell for 1 hour at room temperature (RT) followed by 5 times of washingin PBS+0.05% Tween 20. 50 μl of apex-directed bNAbs in blocking bufferwere added, with a maximum concentration of 2 μg/ml and a 5-folddilution series, and incubated for 1 hour at RT. After washing 5 timesin PBS+0.05% Tween 20, the wells were incubated with 50 μl ofPeroxidase-AffiniPure Goat Anti-Human IgG antibody (JacksonImmunoResearch Laboratories, Inc) at 1:5000 in PBS+0.05% Tween 20 perwell for 1 h at RT. After washed 5 times in PBS+0.05% Tween 20, thewells were developed using TMB at RT for 5 min and the reaction stoppedwith 2 N sulfuric acid. The readout was measured at a wavelength of 450nm.

Binding Analysis by Biolayer Light Interferometry. The kinetics of HIV-1antigen and nanoparticle binding to bNAbs and non-NAbs was measuredusing an Octet Red96 instrument (fortéBio, Pall Life Sciences). Allassays were performed with agitation set to 1000 rpm in fortéBIO 1×kinetic buffer. The final volume for all the solutions was 200 μl/well.Assays were performed at 30° C. in solid black 96-well plates (GeigerBio-One). 5 μg/ml of protein in 1× kinetic buffer was used to load anantibody on the surface of anti-human Fc Capture Biosensors (AHC) for300 s. A 60 s biosensor baseline step was applied prior to the analysisof the association of the antibody on the biosensor to the Env trimer insolution for 200 s. A two-fold concentration gradient of testingantigens was used in a titration series of six. The dissociation of theinteraction was followed for 300 s. Correction of baseline drift wasperformed by subtracting the averaged shift recorded for a sensor loadedwith antibody but not incubated with trimer, or a sensor withoutantibody but incubated with trimer. Octet data were processed byfortéBio's data acquisition software v.8.1. Experimental data werefitted for V1V2 apex-directed bNAbs using a global fit 1:1 model todetermine the K_(D) values and other kinetic parameters.

Example 15 Further Modification of the Gp41 Domain to Improve TrimerProperties

We hypothesize that other regions within gp41 also contribute to theHIV-1 Env metastability, in addition to the N-terminal bend of theheptad region 1 (HR1), which is the primary cause of metastability. Thisis the master hypothesis that can be realized in two differentimplementations.

In the first implementation, we hypothesize that the BG505 gp41 domaincontaining a redesigned HR1 N-terminal bend and a cleavage-site linkercan be used to replace the gp41 domain of diverse HIV-1 strains orsubtypes to render “chimeric” gp140 trimers (termed “UFO-BG”) with thesame high yield, purity, and stability as the BG505 UFO gp140 trimer.Since the gp120 domain encodes most of the Env epitopes and thus definesthe identity of an HIV-1 strain or subtype, such a “chimeric” gp140trimer will be antigenically similar to the wild-type (WT) gp140 trimerbut with significantly improved yield, purity, and stability. Tovalidate this hypothesis, we selected 10 HIV-1 strains of 5 diverseclades (A, B, C, B′/C, and A/E), covering the majority of thecirculating HIV-1 isolates in different geographic regions around theglobe. These 10 HIV-1 strains include BG505 (clade A, tier 2), Q842-d12(clade A, tier 2), 6240.08.TA5.4622 (clade B, tier 2), H078.14 (clade B,tier 3), Du172.17 (clade C, tier 2), 16055-2.3 (clade C, tier 2), CN54(clade B′/C, tier unknown), CH115.12 (clade B′/C, tier 3), 95TNIH022(clade A/E, tier unknown), and 93JP_NH1 (clade A/E, tier unknown).Indeed, the profiles obtained from size-exclusion chromatography (SEC)on a Superdex 200 16/600 Hi-Load column demonstrated exceptional purityfor the UFO-BG trimers derived from diverse HIV-1 strains. Usingbio-layer interferometry (BLI), we further measured the antigenicprofiles for these 10 UFO-BG trimers against a panel of 11 broadlyneutralizing antibodies (bNAbs) and 8 non-NAbs, showing broadly similarpatterns to the wild-type (WT) gp140 trimers without swapping the gp41domain. Negative-stain EM analysis of 10 UFO-BG trimers indicated that80-100% of the produced Env protein corresponds to native-like gp140trimers. Finally, a crystal structure has been determined for the UFO-BGgp140 trimer of a clade-B, tier-3 strain, H078.14, confirming thestructural integrity of such chimeric gp140 designs.

In the second implementation, we hypothesize that a “universal” (or“consensus”) gp41 domain derived from the HIV-1 sequence databasecontaining a redesigned HR1 N-terminal bend and a cleavage-site linkercan be used to replace the gp41 domain of diverse HIV-1 strains orsubtypes to render “chimeric” gp140 trimers (termed “UFO-U”) withimproved yield, purity, and antigenicity. Such universal (or consensus)gp41 can be calculated from the database of all HIV-1 sequence(UFO-UALL), or from the database of HIV-1 sequences belonging to aspecific HIV-1 subtype (for example, UFO-UA, -UB, -UC, -UBC, and -UAE).These databases and their use for obtaining “consensus” sequences arewell known in the art. See, e.g., Kothe et al., Virol. 352:438-449,2006; and Korber et al., Brit. Med. Bullet. 58:19-42, 2001. Todemonstrate the effectiveness of this design strategy, we selected 5representative HIV-1 strains, each from a different clade, tocharacterize the UFO-UALL trimers. Similar to the UFO-BG trimers, weobserved improved SEC profiles and BLI antibody binding profiles for theUFO-UALL trimers compared to the wild-type (WT) gp140 trimers withoutswapping the gp41 domain. Negative-stain EM analysis of the selectedUFO-UALL trimers indicated that 90-100% of the produced Env proteincorresponds to native-like gp140 trimers.

Example 16 Displaying UFO Gp140 Trimers on Bacteriophage Q_(β)Virus-Like Particle

Bacteriophage Q_(β) is an icosahedral virus with a diameter of 25 nmthat can infect Escherichia coli (E. coli). The virus-like particle(VLP) derived from Q_(β) has been used as a multivalent carrier todisplay foreign antigens in vaccine development against influenza (PhaseI), allergic rhinitis and asthma (Phase II, NCT00890734), malignantmelanoma (Phase II, NCT00651703), Alzheimer's disease (Phase II,NCT01097096), hypertension (NCT00500786), nicotine addiction (Phase II,NCT01280968), and type II diabetes mellitus (Phase I, NCT00924105).Clinical trials have demonstrated its safety and immunogenicity inhumans. The Q_(β) VLP is made of 180 identical subunits and can inprinciple display 60 HIV-1 gp41 trimers. However, structural modelingsuggested that there might not be enough space on the VLP surface toaccommodate 60 gp140 trimers. To reduce the surface density, weengineered the Q_(β) VLP by covalently linking two Q_(β) subunits with ashort glycine-serine (GS) linker. We further removed an inward-facingglycosylation site that may affect the assembly of Q_(β) VLP inmammalian cells. A trimeric foldon of the T4 phagehead fibritin (PDBIdentifiers: 4NCV, 4NCW, 4NCU, 1RFO) was then included as a “neck” toconnect the UFO gp140 and engineered Q_(β) subunit. The resultingconstruct, when expressed in mammalian cells, assembled into stable VLPsdisplaying 30 UFO gp140 trimers on each particle.

The invention thus has been disclosed broadly and illustrated inreference to representative embodiments described above. It isunderstood that various modifications can be made to the presentinvention without departing from the spirit and scope thereof.

It is further noted that all publications, patents and patentapplications cited herein are hereby expressly incorporated by referencein their entirety and for all purposes as if each is individually sodenoted. Definitions that are contained in text incorporated byreference are excluded to the extent that they contradict definitions inthis disclosure.

What is claimed is:
 1. A HIV-1 vaccine composition, comprising an HIV-1Env-derived trimer immunogen presented on a self-assembling nanoparticleor a virus-like particle (VLP), wherein the HIV-1 Env-derived trimerimmunogen is a modified gp140 protein comprising a gp120 polypeptide anda gp41 polypeptide, wherein amino acid residues 548-568 of theN-terminus of heptad 1 region (HR1) of the gp41 polypeptide is replacedwith a loop sequence of 6 to 14 amino acid residues in length thatstabilizes the pre-fusion gp140 structure, wherein the numbering of theamino acid residues corresponds to HxB2 nomenclature.
 2. The HIV-1vaccine composition of claim 1, wherein the self-assembling nanoparticlecomprises dihydrolipoyl acyltransferase (E2P), ferritin, or lumazinesynthase (LS).
 3. The HIV-1 vaccine composition of claim 1, wherein thegp41 polypeptide is gp41_(ECTO).
 4. The HIV-1 vaccine composition ofclaim 1, wherein the gp120 and gp41 polypeptides are from differentHIV-1 strains.
 5. The HIV-1 vaccine composition of claim 1, wherein themodified HIV-1 gp140 protein is derived from HIV-1 strain BG505.
 6. TheHIV-1 vaccine composition of claim 1, wherein the loop sequencecomprises (GS)n (SEQ ID NO:23), wherein n is any integer between 3 and7, inclusive.
 7. The HIV-1 vaccine composition of claim 1, wherein theloop sequence comprises (GS)₄ (SEQ ID NO:24).
 8. The HIV-1 vaccinecomposition of claim 1, wherein the loop sequence comprises 10 aminoacid residues.
 9. The HIV-1 vaccine composition of claim 8, wherein theloop sequence comprises any one of SEQ ID NOs:1-5.
 10. The HIV-1 vaccinecomposition of claim 1, wherein the loop sequence comprises 8 amino acidresidues.
 11. The HIV-1 vaccine composition of claim 10, wherein theloop sequence comprises any one of SEQ ID NOs:6-10.
 12. The HIV-1vaccine composition of claim 1, wherein the modified HIV-1 envelopegp140 protein further comprises a flexible linker sequence thatsubstitutes for the cleavage site sequence between gp120 and gp41. 13.The HIV-1 vaccine composition of claim 12, wherein the linker sequencecomprises (G₄S)₂ (SEQ ID NO:22) or SGS and substitutes for residues508-511 at the cleavage site.
 14. The HIV-1 vaccine composition of claim12, wherein the linker sequence comprises 8 amino acid residues andsubstitutes for residues 501-518 at the cleavage site, and whereinnumbering of the amino acid residues corresponds to that of HIV-1 strainBG505. SOSIP.664 gp140.
 15. The HIV-1 vaccine composition of claim 14,wherein the linker sequence comprises the sequence shown in any one ofSEQ ID NOs:16-20.
 16. The HIV-1 vaccine composition of claim 1, whereinthe modified HIV-1 envelope gp140 protein further comprises anengineered disulfide bond between gp120 and gp41.
 17. The HIV-1 vaccinecomposition of claim 16, wherein the engineered disulfide bond isbetween residues A501C and T605C.
 18. The HIV-1 vaccine composition ofclaim 1, wherein the gp41_(ECTO) polypeptide is derived from HIV-1strain BG505, and wherein the N-terminus of heptad 1 region (HR1) (SEQID NO:28) in the gp41_(ECTO) polypeptide is replaced with a loopsequence shown in SEQ ID NO:6.
 19. The HIV-1 vaccine composition ofclaim 1, wherein the modified HIV-1 envelope gp140 protein furthercomprises (a) a linker sequence (G₄S)₂ (SEQ ID NO:22) that substitutesfor residues 508-511 at the cleavage site, and (b) an engineereddisulfide bond between residues A501C and T605C.
 20. A polynucleotideencoding the subunit sequence of the HIV-1 vaccine composition ofclaim
 1. 21. A method of treating or preventing HIV-1 infection in asubject, comprising administering to the subject a pharmaceuticalcomposition comprising a therapeutically effective amount of the HIV-1vaccine composition of claim 1, thereby treating or preventing HIV-1infection in the subject.